Bio-Rad Experion RNA Analysis Kits User Manual

Download

Experion™ RNA StdSens and HighSens Analysis Kits

Instruction Manual

Catalog #700-7103 #700-7105

Bio-Rad Technical Support

For help and advice regarding products from the Experion™ automated electrophoresis system, please contact the Bio-Rad

Technical Support department, which in the United States is open Monday–Friday, 5:00 AM–5:00 PM, Pacific Time.

Phone: 1-800-4BIORAD (1-800-424-6723)

Fax: 1-510-741-5802

Email: LSG_TechServ_US@bio-rad.com (for U.S. and international customers)

Online technical support and worldwide contact information are available at www.consult.bio-rad.com.

Legal Notices

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including

photocopy, recording, or any information storage or retrieval system, without permission in writing from Bio-Rad Laboratories.

Bio-Rad reserves the right to modify its products and services at any time. This user guide is subject to change without notice.

Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors, or for any damages resulting from the application

or use of this information.

Excel is a trademark of Microsoft Corporation.

The dyes used in Experion kits are manufactured by Molecular Probes, Inc. and are licensed for research use only.

LabChip and the LabChip logo are trademarks of Caliper Life Sciences, Inc. Bio-Rad Laboratories, Inc. is

licensed by Caliper Life Sciences, Inc. to sell products using the LabChip technology for research use only.

These products are licensed under U.S. patents 5,863,753; 5,658,751; 5,436,134; and 5,582,977, and pending

patent applications, and related foreign patents, for internal research and development use only in detecting,

quantitating, and sizing macromolecules, in combination with microfluidics, where internal research and development use

expressly excludes the use of this product for providing medical, diagnostic, or any other testing, analysis, or screening services,

or providing clinical information or clinical analysis, in any event in return for compensation by an unrelated party.

Contents

Chapter 1: Experion™ RNA Analysis Kits.............................................. 1

1.1 Product Description .......................................................... 2

1.2 Kit Components ............................................................. 3

1.3 Storage Conditions ........................................................... 3

1.4 Additional Requirements ....................................................... 4

Chapter 2: Essential Practices ...................................................... 5

2.1 Storing and Preparing Samples and Reagents ...................................... 6

2.2 Priming and Loading the Chip................................................... 6

2.3 Running the Analysis.......................................................... 7

2.4 General Maintenance ......................................................... 7

2.5 Experion Video Tutorials ....................................................... 7

Chapter 3: Experion RNA Assay Procedure ........................................... 9

3.1 Set Up the Electrophoresis Station .............................................. 10

3.2 Equilibrate the Kit Reagents ................................................... 10

3.3 Filter the Gel and Prepare the Gel-Stain Solution ................................... 10

3.4 Prepare the Samples and the RNA Ladder ....................................... 11

3.4.1 Experion RNA StdSens Analysis ............................................. 11

3.4.2 Experion RNA HighSens Analysis ............................................ 11

3.5 Prime the Chip ............................................................. 11

3.6 Load the Chip .............................................................. 12

3.7 Run the RNA Analysis ........................................................ 14

3.8 Clean the Electrodes......................................................... 15

3.9 Evaluate the Run............................................................ 15

Chapter 4: Data Analysis ......................................................... 19

4.1 Viewing Data............................................................... 20

4.1.1 Managing Run Files and Project Folders in the Tree View.......................... 20

4.1.2 General Display Controls .................................................. 21

4.1.3 Electropherogram View.................................................... 21

4.1.4 Gel View ............................................................... 25

4.1.5 Results and Settings ..................................................... 26

4.2 Changing the Fluorescence Intensity Scale ........................................ 26

4.3 Using Results and Settings to View and Annotate Data .............................. 27

4.4 Comparing Data from Different Runs ............................................ 28

4.5 Saving, Exporting, and Printing Data............................................. 29

4.5.1 Saving Data Files ........................................................ 29

4.5.2 Exporting Data Files to Other Applications ..................................... 29

4.5.3 Printing Data Files ........................................................ 29

Chapter 5: Assessing RNA Quantity and Quality ...................................... 31

5.1 RNA Quantitation ........................................................... 32

5.2 RNA Quality Indicator (RQI) .................................................... 32

Chapter 6: Changing Analysis Settings and Parameters ................................ 35

6.1 Manually Setting a Marker..................................................... 36

6.2 Changing Peak Finding Parameters ............................................. 36

6.3 Changing General Settings .................................................... 37

6.4 Baseline Modification ........................................................ 37

6.5 Turning Analysis Off ......................................................... 38

6.6 Manual Peak Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Chapter 7: Troubleshooting ....................................................... 41

7.1 Electrophoresis and Priming Stations ............................................ 42

7.2 Experion RNA Analysis ....................................................... 42

7.3 Contacting Technical Support.................................................. 45

Appendices .................................................................... 47

Appendix A: How the Experion System Works ........................................ 48

Appendix B: Deep Cleaning Procedure .............................................. 53

Appendix C: Glossary ........................................................... 54

Appendix D: Ordering Information .................................................. 56

Experion™ RNA Analysis Kits

Experion Automated Electrophoresis System

1.1 Product Description

The Experion RNA StdSens and HighSens analysis kits are used for RNA analysis with the Experion automated electrophoresis system (Figure 1.1). The Experion system employs LabChip microﬂuidic technology to automate nucleic acid and protein electrophoresis and analysis, integrating separation, detection, and data analysis within a single platform. Using much smaller sample and reagent quantities than standard analysis methods, the Experion automated electrophoresis system can be used both upstream and downstream of a number of nucleic acid and protein applications.

The Experion RNA analysis kits are used to determine total RNA and mRNA integrity, purity, and concentration. The Experion RNA StdSens analysis kit offers analysis at nanogram levels, and the Experion RNA HighSens analysis kit is used for analysis at picogram levels (Table 1.1). Both kits include the Experion RNA ladder, which has been optimized for automated electrophoresis on the Experion system. They also feature the Experion RNA loading buffer, which contains a 50 bp marker that is used for the proper alignment of samples to the RNA ladder.

RNA analysis with the Experion system compares favorably with other methods for RNA quantitation, such as UV spectroscopy, and provides analysis of both RNA integrity and concentration in a format that is versatile and easy to analyze. In addition, Experion analysis is relatively unaffected by reagents common to RNA preparation, and the electropherogram it generates enables evaluation of the RNA sample for degradation and for the presence of copurifying nucleic acids, such as genomic DNA and tRNA (Woo and Strong 2006). The Experion RNA StdSens and HighSens analysis kits offer single-step total RNA and mRNA analysis in less than 30 min.

For details about how the Experion RNA analysis kits analyze RNA, refer to Appendix A in this manual.

Register your Experion system to ensure you receive important updates on software, tech notes, and manuals. Upon installation, a dialog provides registration instructions.

Fig. 1.1. The Experion system. The system

includes the following components: 1) automated electrophoresis station, 2) priming station,

3) vortex station used for nucleic acid analysis only, 4) system ope ration and data analysis tools (software), and 5) analysis kits, which include the (a) chips and (b) reagents for protein (Pro260 kit),

standard-sensitivit y RNA (StdSens kit), high-sensitivity RNA (HighSens kit), and DNA (DNA 1K and 12K kits) analyses.

2 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

Table 1.1. Experion RNA analysis kit speciﬁcations.

Experion RNA StdSens Experion RNA HighSens

Number of samples 12 11

Sample volume 1 µl 1 µl

Quantitation range:

Total RNA 25–500 ng/µl 200–5,000 pg/µl mRNA 25–250 ng/µl —

Limit of detection (total RNA) 5 ng/µl 100 pg/µl

Maximum salt concentration TE buffer (10 mM Tris, 1 mM EDTA) DEPC-treated water

1.2 Kit Components

Table 1.2. Components of the Experion RNA analysis kits.

Item Description Volume (per Vial) 10-Chip Kit

RNA StdSens or Microﬂuidic chips used for RNA separation 10 chips RNA HighSens chip

Cleaning chip Chip used for cleaning electrodes 2 chips

RNA gel Proprietary polymeric sieving matrix 1,250 µl 1 vial

RNA StdSens or Proprietary ﬂuorescent dye 20 µl 1 vial RNA HighSens stain

RNA StdSens or Buffer for sample preparation; contains lower marker 900 µl 1 vial RNA HighSens loading buffer for alignment of samples to the RNA ladder

RNA ladder Standard containing 8 RNA fragments of 200 –6,000 nt 20 µl 1 vial

RNA sensitivity enhancer Proprietary reagent to improve sensitivity 100 µl 1 vial (RNA HighSens kit only)

Spin ﬁlters Used for ﬁltering reagents during sample preparation 3 ﬁlters

1.3 Storage Conditions

Table 1.3. Storage conditions.

Item Storage Shelf Life

Experion reagents 4ºC See expiration date on packaging

Experion chips Ambient See expiration date on packaging

Experion RNA ladder –70ºC See expiration date on packaging

Filtered gel (G) 4ºC 1 month from ﬁltration; after 1 month, reﬁlter

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 3

Experion Automated Electrophoresis System

1.4 Additional Requirements

Experion automated electrophoresis station

Experion priming station

Experion vortex station

Microcentrifuge (1,000–10,000 x g)

Benchtop vortexer

Aluminum foil

Calibrated pipets and RNase-free, narrow-bore barrier tips (for example, VWR #87001-688 or Rainin #L-10F)

RNase-free microcentrifuge tubes, 0.5 ml or 0.65 ml

RNase-free water (for example, Experion DEPC-treated water, catalog #700-7253)

Benchtop centrifuge (optional, catalog #166-0612)

Extra spin ﬁlter for additional ﬁltration steps (as needed, catalog #700-7254)

Experion electrode cleaner (catalog #700-7252)

Foam cleaning swabs (catalog #700-7264)

4 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Essential Practices

Experion Automated Electrophoresis System

2.1 Storing and Preparing Samples and Reagents

Store all Experion™ reagents except the RNA ladder at 4°C when not in use. Store the RNA ladder at –70ºC. Do not store reagents at room temperature for >2 hr, as this will shorten their shelf life.

Before use, allow all kit reagents (except the RNA ladder) to equilibrate to room temperature (15–20 min). Once thawed, gently vortex all kit reagents before use. Before opening the tubes, quickly centrifuge them to collect solution to the bottoms of tubes.

If the RNA gel has frozen, discard it.

Protect the RNA stain and gel-stain solution (GS) from light: store these solutions in a dark place and keep them covered with foil when using them.

The RNA stain contains DMSO, which is hygroscopic. Cap tightly.

Prepare GS on the day of use. Use ﬁltered gel (G) for up to 1 month. After 1 month, reﬁlter G.

Do not use coated or treated pipet tips or microcentrifuge tubes (for example, siliconized polypropylene) for preparation of kit reagents or samples. Use of treated tips or tubes may cause separation artifacts.

Use RNase-free microcentrifuge tubes, pipet tips, and TE buffer for sample and reagent preparation.

2.2 Priming and Loading the Chip

To avoid contamination, wear gloves and handle chips by the edges. Never touch the glass portions of the chip.

Load the chip on a benchtop or in the priming station. Never load a chip in the electrophoresis station.

Avoid sources of dust and other contaminants when preparing samples and loading the chip. Foreign particles in reagents, samples, or the wells of the chip interfere with separation. Remove chips from their packaging immediately before use.

It may be easier to load the chip on a white background. Tilt the chip to look for bubbles.

Use narrow-bore ﬁlter pipet tips for loading the chip (for example, VWR #87001-688 or Rainin #L-10F).

To avoid introducing air bubbles, do the following (for more help with chip loading, refer to the Experion Training Video in the Experion software Help section under Contents and Index > Contents > Appendices > Technical Videos):

Insert the pipet tip all the way to the bottom of the chip well when dispensing liquids

(this reduces the possibility of trapping air)

Hold the tip vertically, perpendicular to the chip surface. Holding the tip at an angle

may trap air bubbles at the bottom of the well

When expelling liquid, dispense slowly and only to the ﬁrst stop on the pipet. Using the

second stop introduces air and bubbles into the liquid. Reverse pipetting is acceptable

Dislodge bubbles at the bottom of a well with a clean pipet tip, or remove the solution and load it again.

Use a primed and loaded chip within 5 min of loading. When chips are not used within this time, reagents may evaporate, leading to poor results or a chip performance error.

Fill all the chip wells when running an analysis. Use blank samples (prepared with water instead of sample) or replicates if necessary. All 16 electrode pins must be in contact with liquid; otherwise, an IV (current voltage) check failure error will occur.

6 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

2.3 Running the Analysis

Place the electrophoresis station on a stable surface, where it will not be subjected to vibrations or other movement, and away from direct sunlight and all other potential sources of extreme heat.

Power on the electrophoresis station before launching Experion software.

The ﬁrst time that the Experion electrophoresis station is used, conﬁrm that communication has been established between the software and electrophoresis station before preparing the reagents.

Do not open the lid of the electrophoresis station during a run. The run will abort if the lid is opened.

2.4 General Maintenance

For recommendations on general instrument maintenance, refer to the Experion system manual (bulletin 10001312).

Clean the electrodes after each run (routine cleaning). Cleaning maintains the instrument in optimum condition and prevents buildup and cross-contamination of reagents and samples.

Perform the deep cleaning procedure described in Appendix B to clean the electrodes:

Prior to ﬁrst use of the Experion electrophoresis station

Whenever contamination is suspected or visible (for example, salt deposits or other

precipitates) on the electrodes

Whenever a chip has been left in the electrophoresis station for an extended period of time

(for example, overnight)

Never store the cleaning chip inside the electrophoresis station. Store the empty cleaning chip covered to keep the wells clean. Two new cleaning chips are included with every box of chips.

2.5 Experion Video Tutorials

For additional information, view the video tutorials available online at www.bio-rad.com:

North America: Home > Life Science Research > Support > Tutorials > Electrophoresis and Blotting > Experion System Training

Other: Home > Life Science Research > Electrophoresis > Automated Electrophoresis > Experion Training Videos

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 7

Experion Automated Electrophoresis System

8 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion™ RNA Assay

Procedure

For an abbreviated version of this protocol, refer to the Quick Guide provided with the kit.

Experion Automated Electrophoresis System

3.1 Set Up the Electrophoresis Station

1. If needed, perform a deep cleaning of the electrodes (see Appendix B for instructions).

2. Power on the computer and then power on the Experion electrophoresis station by pushing the green button in the center of the front panel. The steady green LED above the button indicates the unit is on.

3. Launch Experion software. If the instrument and computer are communicating properly:

A green dot and the last 4 digits of the instrument serial number appear at the lower right of the

software screen

The electrophoresis station icon appears in the upper left corner

When there is no connection, these indicators are absent and a grayed-out instrument icon appears at the upper left of the software screen.

3.2 Equilibrate the Kit Reagents

1. Set a heating block or water bath to 70°C. You will use this heating device to denature the samples and the RNA ladder later in the protocol.

2. Thaw the following kit components on ice (15–20 min):

RNA samples

RNA ladder (red cap)

3. Equilibrate the following kit components to room temperature (15–20 min):

RNA stain (blue cap)

RNA loading buffer (yellow cap)

RNA gel (green cap) (if ﬁltered gel was prepared previously, remove it from storage and

equilibrate to room temperature)

Sensitivity enhancer (clear cap, RNA HighSens only)

4. Invert each tube several times and then vortex to reincorporate any condensed liquid. Brieﬂy centrifuge the solutions to the bottom of the tubes. Make sure the RNA stain solution (blue cap) is thawed before proceeding.

3.3 Filter the Gel and Prepare the Gel-Stain Solution

A gel-stain solution (GS) preparation is sufﬁcient for use with up to three RNA chips. Prepare GS daily and keep it covered with foil until ready for use. If ﬁltered gel (G) is available, skip step 1. Use G within 1 month of preparation. After 1 month, reﬁlter it before reuse.

1. Pipet 600 μl RNA gel (green cap) into a spin ﬁlter and centrifuge it at 1,500 x g for 10 min. Inspect the tube to ensure all of the gel has passed through the ﬁlter and then discard the ﬁlter.

2. Prepare the GS. Pipet 65 μl G into an RNase-free 0.65 ml microcentrifuge tube, add 1 μl RNA stain, and vortex for 10 sec. Wrap the tube of GS in aluminum foil to protect the stain from light.

3. RNA HighSens analysis only: Centrifuge the GS at 13,000 x g for 10 min.

10 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

3.4 Prepare the Samples and the RNA Ladder

Once the RNA samples and RNA ladder (red cap) have thawed on ice, vortex them brieﬂy and spin down for a few sec in a microcentrifuge. Both the sample and RNA ladder must be denatured. Use of the RNA HighSens ladder also requires dilution.

3.4.1 Experion RNA StdSens Analysis

1. Pipet ≥2 μl RNA sample into separate RNase-free microcentrifuge tubes.

2. Pipet RNA ladder into an RNase-free microcentrifuge tube: use 1 μl RNA ladder for each chip plus an extra 1 μl RNA ladder to accommodate variations in pipetting (for example, when running 1 chip, use a total of 2 μl RNA ladder; for 3 chips, use 4 μl RNA ladder).

3. Denature the samples and the RNA ladder by heating the tubes at 70°C for 2 min. Place them on ice for 2–5 min.

4. Vortex brieﬂy and spin down. Keep the tubes on ice.

3.4.2 Experion RNA HighSens Analysis

If diluted RNA ladder is available from a previous preparation, skip steps 1 to 5. Do not reheat the diluted ladder.

1. Pipet 5 μl RNA ladder into an RNase-free microcentrifuge tube.

2. Transfer 2 μl RNA sample into separate RNase-free microcentrifuge tubes.

3. Denature the samples and the RNA ladder by heating the tubes at 70°C for 2 min. Place them on ice for 2–5 min.

4. Spin down the ladder and samples in a microcentrifuge brieﬂy and then place the tube on ice. Add 795 μl DEPC-treated water to the denatured ladder. Keep the tubes on ice.

5. Aliquot diluted RNA ladder into RNase-free microcentrifuge tubes, store at –70°C, and avoid exposing it to freeze-thaw cycles.

3.5 Prime the Chip

Start the run within 5 min of priming and loading the chip. For help with chip loading, refer to the Experion Training Video: Chip Loading, available in the Experion software Help section under Contents and Index > Contents > Appendices > Technical Videos.

1. Pipet 9 µl GS into the highlighted well labeled GS (gel priming well, Figure 3.1).

2. On the priming station, set the pressure setting to B and the time setting to 1, as speciﬁed by the alphanumeric code on the chip (Figure 3.1).

3. Open the Experion priming station and place the chip on the chip platform, matching the arrow on the chip with the alignment arrow on the chip platform. A post on the chip prevents insertion in the wrong position. Do not force the chip into position.

4. Close the priming station by pressing down on the lid. The lid should snap closed.

5. Press Start. A “Priming” message appears on the screen of the priming station, and the timer counts down. Priming requires ~30 sec. Do not open the priming station during the countdown.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 11

Experion Automated Electrophoresis System

Gel priming well

Priming code

Fig. 3.1. Experion RNA chips. The locations of the gel priming well (GS, highlighted) and alphanumeric priming codes are indicated.

Gel priming well

Priming code

6. An audible signal and “Ready” message indicate that priming is complete. Open the priming station and remove the chip. If the lid sticks, press down on it while pressing down on the release lever.

7. Turn the chip over and inspect the microchannels for bubbles or evidence of incomplete priming. If the chip is primed properly, the microchannels are difﬁcult to see (compare a primed chip to a new, unused chip). If you detect a problem, such as a bubble or incomplete priming, prime a new chip.

8. Place the chip on a clean surface for loading.

Bubbles forced into microchannels during priming take the shape of the microchannel and are elongated, not round.

3.6 Load the Chip

1. Pipet 9 µl GS into the other well labeled GS (Figure 3.2).

2. Pipet 9 µl ﬁltered gel (G) into the well labeled G.

(HighSens only) Pipet 6 µl sensitivity enhancer (clear cap) into the well labeled SE.

4. Pipet 5 µl RNA loading buffer (yellow cap) into each sample well (HighSens wells 1–11 or StdSens wells 1–12, ) and the ladder well, labeled L.

Use a new pipet tip for each delivery to prevent contamination of the loading buffer. Alternatively, aliquot 65–70 μl loading buffer into an RNase-free microcentrifuge tube and add 5 μl to each well from this volume.

5. Pipet 1 µl prepared RNA ladder into the well labeled L. Every chip must have the RNA ladder loaded into the ladder well labeled L.

6. Pipet 1 µl sample (or blanks, for example loading buffer, DEPC-treated water, or TE buffer) into the numbered sample wells.

12 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

7. Inspect all wells for bubbles by holding the chip above a light-colored background and looking through the wells (Figure 3.3). Dislodge any bubbles at the bottom of a well with a clean pipet tip or by removing and reloading the solution.

Load 5 µl loading buffer into sample wells and well labeled L

Load 1 µl sample into wells 1 –11

Load 1 µl ladder into well labeled L

Fig. 3.2. Experion RNA HighSens chip. Wells for loading GS, s amples, and ladder are indicated.

Load 9 µl G into well labeled G

Load 9 µl GS into other well labeled GS

HighSens only: load 6 µl sensitivity enhancer into well labeled SE

Fig. 3.3. Bubble formation during loading of Experion Pro260 chips. Surface bubbles do not generally cause problems during a run, but bubbles at the bottoms of wells must be removed. Left, bubbles trapped at the bottom of wells. The GS and G wells and sample wells 1, 3, and 4–6 contain no solution. Wells 8, 10, and L are ﬁlled properly and have no bubbles, but large bubbles have formed at the bottoms of wells 7 and 9 (note the difference in the diameter of the light-colored circles in wells 8 and 9). Right, bubbles have formed at the surface of the three GS wells on the right side of the chip; the rest of the wells have no bubbles.

8. Slide the chip into the Experion vortex station and turn on the vortex station by pressing Mix. Vortexing continues for 60 sec and then automatically shuts off. Remove the chip when the vortex station stops.

9. Inspect the wells again to conﬁrm that there is no excessive bubble formation from pipetting and that no liquid has spilled outside the wells during vortexing.

10. Place the loaded chip into the Experion electrophoresis station and start the run within 5 min.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 13

Experion Automated Electrophoresis System

3.7 Run the RNA Analysis

1. Open the lid of the electrophoresis station by pulling the release latch. Place the primed, loaded, and vortexed chip on the chip platform and close the lid.

2. In the Experion software toolbar, click New Run . In the New Run screen (Figure 3.4), from the Assay pull-down list, select the type of assay.

3. Either select a project folder from the Project pull-down list or create a new project folder by entering a name in the Project ﬁeld or by selecting File > Project > New. The project folder appears in the project tree.

4. Enter a name for the run in the Run Preﬁx ﬁeld and click Start Run .

Fig. 3.4. Details of the New Run scre en. The green dot in the lower right corner indicates that communication between the electrophoresis station and Experion software has been established.

5. In the New Run dialog (Figure 3.5), select the number of samples to be analyzed. Though all wells are ﬁlled, the Experion system stops the analysis when it reaches the number of samples entered.

Fig. 3.5. New Run dialog. The Experion system stops analysis when it reaches the number of samples entered.

14 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

6. Click Start. The green LED in the center of the front panel on the electrophoresis station blinks, and the system performs a number of checks: it conﬁrms that a chip has been inserted, that all wells contain liquid, that electrical connections are made, etc. A calibration counter marks the progress of these calibrations at the upper right of the screen.

Do not open the lid of the Experion electrophoresis station until the run is complete. The lid does

not lock. Opening the lid aborts the run.

An “IV Check Error” message indicates the system cannot make electrical contact in one of the wells. This often means there is a bubble at the bottom of the well. Abort the run, and check the chip for bubbles or empty wells. Reﬁll the affected well(s), and start the run again.

7. During separation, the sample name is highlighted in the project tree and the electropherogram trace, and virtual gel bands appear in real time:

The electropherogram of the sample being separated appears in the electropherogram

view

The lane corresponding to that sample is outlined in pink and has a dark background

To display the electropherogram of another sample, click on either the sample name in the project

tree or on a lane in the virtual gel.

8. When analysis is complete (after ~30 min), the instrument beeps and a window opens indicating the end of the run. Select OK and remove the chip from the chip platform.

9. Clean the electrodes using deionized water within 30 min of each run.

3.8 Clean the Electrodes

1. Fill a cleaning chip with 800 µl deionized water (0.2 µm-ﬁltered). Gently tap the side of the cleaning chip to remove any trapped bubbles from the wells.

2. Place the cleaning chip on the chip platform in the electrophoresis station, close the lid, and leave it closed for 1 min.

Never store the cleaning chip inside the electrophoresis station. Store the empty cleaning chip covered to keep the wells clean. Two cleaning chips are included with each box of chips.

3. Open the lid, remove the cleaning chip, and allow the electrodes to dry for 1 min. Close the lid.

4. Replace the water in the cleaning chip after use to avoid contamination. For storage, remove the water from the cleaning chip and store the chip in a clean location.

3.9 Evaluate the Run

When a run is complete, evaluate the run and the analysis of the data by Experion software.

1. Ensure that all lanes (ladder and samples) are visible in the virtual gel. The lower marker (indicated by a pink triangle) should be visible in and aligned across all lanes. If the marker peak is not properly assigned, you may need to manually set the marker (see Section 6.1).

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 15

Experion Automated Electrophoresis System

2. Evaluate the separation of the RNA ladder. To display the ladder electropherogram, click the ladder well in the project tree, or click on the lane labeled L in the virtual gel. The electropherogram should resemble the one shown in Figure 3.6 and should have the following features (if your ladder does not have these features, see Chapter 7, Troubleshooting for more information):

Lower marker peak at ~20 sec, at least 5 ﬂuorescence units above the baseline, and

distinguishable from the ladder peaks

Eight resolved ladder peaks

Flat baseline

RNA lad der peaks

Lower marker

Baseline

Fig. 3.6. Experion RNA StdSens separ ation of the RNA ladder in a prokar yotic total RNA assay. Note the ﬂat baseline and resolved peaks.

3. Examine the separation of at least one sample. Click on the sample name in the project tree or on the lane in the virtual gel to view the electropherogram, which should have the following features:

Lower marker peak at ~20 sec, at least 5 ﬂuorescence units above the baseline, and

distinguishable from the sample peaks

Sample peaks: mRNA analyses often yield broad peaks and, on occasion, contaminating

18S and 28S rRNA peaks (Figure 3.7), while total RNA analyses often yield 2–3 prominent peaks corresponding to rRNA (in eukaryotic sources, 18S and 28S and occasionally 5S; in prokaryotic sources, 16S and 23S) (Figure 3.8)

Flat baseline

If the lower marker peaks are not properly assigned, manually set the marker (see Section 6.1).

4. Evaluate the data analysis performed by Experion software (see Chapter 4, Data Analysis). If necessary, change the analysis settings and parameters by following the instructions in Chapters 5 and 6.

16 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

Fig. 3.7. Experion RNA HighSens analysis of mR NA. Note the contaminating 18S rRNA peak identiﬁe d by Experion sof tware. Analysis time for mRNA samples is longer than for total RNA samples to include the population of longer transcripts and to achieve a smooth baseline.

Fig. 3.8. Experion RNA HighSens analysis of eukar yotic total RNA . The 18S and 28S rRNA peaks are identiﬁed by Experion software.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 17

Experion Automated Electrophoresis System

18 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

This chapter outlines the basic steps and software features used to view and analyze data. For a more detailed description of Experion™ software and its various functions, refer to the software Help menu. For information on how to customize analysis parameters, refer to Chapter 6, Changing Analysis Settings and Parameters.

Data Analysis

Experion Automated Electrophoresis System

4.1 Viewing Data

The main window of the Experion software user interface contains menus and toolbars, a tree view, and three data views: the electropherogram view, gel view, and results and settings tabs. (Figure 4.1).

Menu ba r

Main sc reen tool bar

Electr ophero gram view

Tree view

Displ ay toolbar

Gel vie w

Resul ts and set tings tabs

Fig. 4.1. Main window of Experion software. The toolbars, menus, and data views are indicated.

4.1.1 Managing Run Files and Project Folders in the Tree View

The tree view (Figure 4.2) displays the project tree, a hierarchical tree of all projects and the data ﬁles they contain. Use the project tree and the toolbar above it to:

Open run ﬁles and select samples for viewing

Create project folders

Export and import one or more run ﬁles

Edit names of run ﬁles and project folders

Move runs into project folders

Delete run ﬁles and project folders

Toolbar

Projec t folder

Run ﬁle

Sample

Fig. 4.2. Tree view details. The proje ct folder name, sample well label, and run ﬁle preﬁx can be customize d.

20 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

To open a run ﬁle, double-click on it. The run ﬁle opens, displaying the electropherogram and Results tab for the RNA ladder, and the virtual gel for the entire run.

To view the data from a sample, click on the sample name or select the corresponding lane in the gel view.

To edit a run ﬁle or project folder name, right-click on it and select Rename Project or Rename Run. Type in the new name.

To import, export, move, or delete ﬁles and folders, click the icons in the toolbar or use the context menu accessed by right-clicking on the items in the tree view. For more details, refer to the Experion software Help ﬁle.

4.1.2 General Display Controls

Data in the electropherogram view, gel view, and results and settings tabs are linked. Selecting a sample or peak in one area automatically opens or highlights that sample/peak in the other two areas.

To adjust the relative sizes of the three data views and the tree view, click on and drag the border of the view to its new location.

To hide or show one of the three data views, click on the corresponding icon in the display toolbar (located in the upper-right corner of the display, just above the gel view, Figure 4.1).

To zoom in on (expand the view of) a portion of an electropherogram or virtual gel, click on the corner of the area that you would like to enlarge and mouse over to the opposite corner. Double-click anywhere in the electropherogram view to return to the previous view.

4.1.3 Electropherogram View

Experion software plots ﬂuorescence intensity vs. migration time to generate an electropherogram for each sample.

Place the cursor over a peak or area of an electropherogram to view its calculated size (Figure 4.3).

Fig. 4.3 . Example of an electropherogram from an RNA StdSens analysis of a tota l RNA sample.

Electropherogram displayed with the Results tab selected. Place the cursor over a peak to reveal its size.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 21

Experion Automated Electrophoresis System

To view the peaks identiﬁed by Experion software, click Peaks in the results and settings tabs area. In the electropherogram, all peaks identiﬁed by Experion software become numbered, and the numbers appear under the peaks (Figure 4.4). Peaks generated by the lower marker used for normalization are numbered and labeled in green with an asterisk (*).

Fig. 4.4. Example of an electropherogram from an RNA StdSens analysis of a total RNA sample.

Electropherogram displayed with the Peaks tab selected.

Double- or right-click on a peak to select it. An inverted black arrow appears above the peak in the electropherogram, and a pink arrow appears above the corresponding band in the virtual gel. The peak number and corresponding data also appear highlighted in the Peaks tab.

Display electropherograms in three different ways:

One at a time (single-well view)

All at once in separate windows (all-wells view)

As superimposed images (overlays)

Single-Well View

This is the default view, which shows the electropherogram data from a single well (Figure 4.5). To return to this display:

1. Click on the sample in the project tree or on the corresponding lane in the virtual gel.

2. In the main screen toolbar, click View Single Well or select View > Single Well.

3. The electropherogram appears. The corresponding lane in the virtual gel is outlined in pink.

22 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

Fig. 4.5. Single-well view. As indicated by the arrows, a single electropherogram appe ars, the name of the sample is highlighted in the project tree, and the corresponding lane in the virtual gel is outlined.

All-Wells View

To display all electropherograms simultaneously (Figure 4.6):

1. In the main screen toolbar, click View All Wells or select View > All Wells.

2. Electropherograms for each sample populate the electropherogram view (Figure 4.6). Use the

scroll bar to scroll through the electropherograms, or minimize the results and settings table to accommodate the multiple electropherograms.

3. To select and expand a particular electropherogram, double-click on it. The data appear in a

single-well view.

Fig. 4.6. All-wells view.

All electropherograms are shown in the electropherogram view. Use the scroll bar to view electropherograms.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 23

Experion Automated Electrophoresis System

Electropherogram Overlays

Superimpose (overlay) multiple electropherograms to facilitate direct comparison among proﬁles. To overlay a subset of electropherograms (Figure 4.7), use one of the following options:

Move into the single-well view and, in the main screen toolbar, click Start Overlay .

Select wells (lanes) in the virtual gel. Each gel lane that is displayed in the electropherogram is outlined. To end the overlay, click End Overlay

Select the lanes in the virtual gel while holding the Ctrl key (for noncontiguous, individual

lanes) or the Shift key (for lanes next to the original lane selected). To exit, click on a lane in the virtual gel or on a sample name in the project tree

Select Analysis > Start Overlay. Select the lanes to overlay. To end the overlay, select

Analysis > End Overlay

Electropherograms appear superimposed in a single window, each in a different color. (The colors cannot be changed.) The sample name appears at the top of the electropherogram, in a color that corresponds to the trace. The ﬂuorescence scale of the overlay electropherogram is based on the sample that is selected ﬁrst; therefore, select the sample with the highest peaks ﬁrst.

Fig. 4.7. Electr opherogram overlay. Electropherograms are

shown in different colors, and the corresponding lanes in the virtual gel are outlined. In this example, lanes 5, 7, and 9 are superimposed using the overlay feature.

Peak Labels

To change the type of label displayed, select Graph > Peak Info (or use the pull-down list in the main screen toolbar) and select one of the following options:

No Peak Info — displays no peak information

Peak Number — uses sequential numbers for peak identiﬁcation (default selection)

Peak Time — uses peak migration time (min:sec) for peak labels

Peak Height — uses peak height in units of ﬂuorescence intensity for peak labels

Peak Cor. Area — uses calculated (corrected) peak area for peak labels

Electropherogram Tags (Annotations)

To annotate a peak in the electropherogram, right-click on the peak and select Add Tag (Figure 4.8). A tag is automatically generated with the peak information selected (for example, peak number).

To edit a tag, right-click on it, select Edit Tag, and change the information. Click

OK.

24 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

To annotate a region in the electropherogram, right-click in the region and select Add Tag. In the Edit Tag dialog, enter the annotation and click OK.

To move a tag, click and drag the label to the desired place in the electropherogram.

Fig. 4.8 . Tagged (annotated) peaks in a n electropherogram.

4.1.4 Gel View

Experion software converts electropherogram data into densitometric bands, which appear in the virtual gel (Figure 4.9). Each lane of the virtual gel corresponds to a different sample, and all samples from a chip are shown in a single gel view. When a sample is selected for view as an electropherogram, the corresponding lane in the virtual gel is outlined in pink.

Right-click on a band to select it. A pink arrow appears above the selected band in the virtual gel, and an inverted black arrow appears above the corresponding peak in the electropherogram. The peak number and corresponding data also appear highlighted in the Peaks table. Only one peak can be selected at a time.

To select a different color scheme, choose View > Gel Color and select a color from the list. By default, the gel view displays bands as black signals on a white background.

To change the contrast of the bands, use the sliding cursor (Figure 4.9). Changing the contrast does not change the data, but it may improve visualization of faint bands in the virtual gel.

Sliding curso r

Fig. 4.9. Gel view. Move the sliding cursor to adjust band intensity.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 25

Experion Automated Electrophoresis System

4.1.5 Results and Settings

The six results and settings tabs (Figure 4.10) present options for viewing, customizing, and analyzing separation data (see Section 4.3, Using Results and Settings to View and Annotate Data).

Fig. 4.10. Results and settings. Shown is the Results tab for a single sample.

4.2 Changing the Fluorescence Intensity Scale

To display more or less detail in the electropherogram traces and lanes in the virtual gel, use the options available under the Graph menu. These options determine the y-axis scale (ﬂuorescence intensity) of electropherograms and deﬁne the relative scale intensity of the lanes in the virtual gel (Figure 4.11):

Scale To Selected — uses the ﬂuorescence value of the tallest peak in a selected

electropherogram to set the scale of the y-axis for all samples. Use this option when comparing (overlaying) a well with a high-intensity peak and one with a less intense peak

Scale To Global — uses the ﬂuorescence value of the tallest peak from all the

electropherograms to set the scale of the y-axis for all samples. Use this option to create a virtual gel similar to an agarose gel

Scale To Local (default) — uses the ﬂuorescence value of the tallest peak in each

electropherogram to set the scale of the y-axis for that electropherogram: each sample is scaled separately. This is the best way to view electropherograms and sample details

Scale To Selected Peak — uses the ﬂuorescence value of the selected peak to set the

scale of the y-axis for all samples

Scale To Sel ected

Fig. 4.11. Options for modifying the ﬂuorescence intensity scale. E xamples of how each option af fects the display of a virtual gel are shown.

Scale To Glo bal

Scale To Loca l

Scale To Sel ected Pea k

26 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

4.3 Using Results and Settings to View and Annotate Data

The following options appear under the results and settings tabs:

Results — summarizes analysis data for a single sample. For total RNA, results include

peak or fragment name, migration time, area, % of total area, concentration, 28S:18S ratio for the sample, and the RNA quality indicator (RQI). For mRNA, they include an estimation of rRNA contamination and do not include the RQI or 28S:18S ratio. The data in this table can be exported (see Section 4.5.2, Exporting Data Files to Other Applications)

To view the results of a particular sample, click the electropherogram in the all-wells view or lane in the gel view, or select that sample from the project tree. Alternatively, use the arrows in the main screen toolbar to navigate through the samples

Run Summary — displays a run summary for all samples (wells), including data for each

sample name, RNA area and concentration, 28S:18S ratio and RQI information (total RNA) samples, and rRNA contamination (mRNA); right-click to export the data

RNA — summarizes all RNA fragments identiﬁed in all samples and categorizes them by

their identity (for example, 18S; see below for instructions on how to add fragments for analysis)

Peaks — identiﬁes and provides integration information for peaks in a sample. When you

click on the data row for a peak, the row becomes highlighted, an inverted black arrow appears above the corresponding peak in the electropherogram, and a pink arrow appears above the selected band in the virtual gel. To customize the types of data shown, see the instructions below

Run Info — displays sample names and comments; information can be added before,

during, or after a run (see below for instructions)

Settings — used mostly for troubleshooting but can be used to edit the parameters used

to identify peaks (see Section 6.2, Changing Peak Finding Parameters or the Experion software Help ﬁle for more details about these settings)

Experion software automatically detects and names rRNA fragments. To add a fragment to the analysis:

1. Open the Results tab and either click Edit Fragments or right-click on one of the fragments in the

table.

2. In the Edit Fragments dialog, click Add a RNA name to table . This opens a new row.

3. Enter the information for the RNA fragment and click OK.

To customize the types of data shown under the Peaks tab:

1. Click the arrow to the left of a column header (or right-click on a column header) to open the Column

Selector dialog.

2. Use the arrow keys to show, hide, or change the order of display of any of the available headers.

For detailed explanations of each setting, refer to the software Help ﬁle.

3. Click OK to save the changes, or click Cancel to close the dialog without saving the changes.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 27

Experion Automated Electrophoresis System

To enter sample names and descriptions:

1. Click the Run Info tab and then click on the plus sign (+) next to Sample Information.

2. Click on any active cell, enter the sample name and description, and click Apply. Click File/Save to save the changes. The sample name appears both in the tree view and above the electropherogram.

To enter other information about the experiment:

1. In the Run Info tab, click the plus sign (+) next to the Study Information and/or System Information area(s).

2. Double-click in any blank ﬁeld, enter the information, and click Apply. Click File/Save.

4.4 Comparing Data from Different Runs

Experion software allows you to overlay runs from different chips (same assay type), enabling direct comparison of electropherograms and virtual gels from multiple chips. You can save and edit this new virtual chip, but you will not be able to make any new calculations. All calculations in the Results tab carry over from the original runs.

In security mode, all run comparisons are saved and cannot be deleted.

1. Click Create a New Compare Run or select Analysis > New Compare Run. In the Compare Runs Setting dialog, enter a name for the comparison in the Compare Name ﬁeld.

2. Select the project in which you wish to store the comparison from the Target Project pull-down list.

3. Select the assay from the Assay Type pull-down list and click Next. The Compare Runs dialog opens, displaying only runs of the same assay type.

4. Double-click to expand the run ﬁles you want to compare, or select a run ﬁle and click List Samples.

5. Click the run ﬁle or sample(s), and use the arrows to move them to and from the Compare ﬁeld. To select several lanes at a time, click the sample names while holding the Ctrl or Shift key. To open a window displaying the selected samples in a gel view, click Show Gel Lanes.

Experion software accepts up to 40 samples for comparison (the default is 30). To change the number of wells used, select Tools > Options. Under the Advanced tab, change the number under Visible Compare Runs Gel lanes.

Only one ladder can be used per comparison. If a ladder is not selected, the ladder used in the ﬁrst run ﬁle is used as the ladder for the entire virtual chip. If an additional ladder is inserted in the virtual chip, it is treated as a sample.

6. Select Realign Data to align the results of all the runs and click OK. The separations appear together in one window as a single experiment. In the Results table, the sizes and concentrations for fragments in each sample are unchanged; the original sizes and concentrations are shown.

7. To change the lanes used in the run comparison:

a. Open a run comparison by double-clicking the ﬁle name in the tree view and then clicking Edit

Compare Run or selecting Analysis > Edit Compare Run.

28 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

b. Select the samples you want to add or delete, and use the arrows to add or remove them from

the comparison.

c. To view the samples within a run, double-click the run ﬁle or select it and click List Samples.

8. Click OK to process the changes and view the results.

4.5 Saving, Exporting, and Printing Data

4.5.1 Saving Data Files

Data are automatically saved to a run ﬁle. Names for run ﬁles are given according to the selection made in the Run Files tab of the Options dialog.

To designate a new name for a ﬁle, select File > Save As and enter the desired name.

To view the selections or make changes in the automatic naming of ﬁles, select Tools > Options > Run Files. The following options appear:

Create Run name by combining — select the options to be added to the ﬁle name.

Options include the preﬁx (enter the desired characters), assay class (adds the assay type to the ﬁle name), instrument name, date, and time

Run ﬁle folder — choose between default (saves ﬁles to the directory C:\Program Files\

BioRad Laboratories\Experion Software\Data) and custom (enables navigation and selection of another folder)

Create daily folders — select to enable creation of daily subfolders by date. When this

option is selected, the software stores all run ﬁles from the same date in a single folder

4.5.2 Exporting Data Files to Other Applications

To export data to another program (for example, Excel software), select File > Export Data. In the Export dialog, select the desired options. For a complete description of all export options, refer to the

Experion software Help ﬁle.

To export ﬁles automatically after a run is completed:

1. Select Tools > Options and click the Advanced tab.

2. Select Auto Export and then click Settings.

3. Select the options for Auto Export and click Apply.

To quickly copy and paste electropherograms, virtual gels or lanes in the gels, or data tables into another application, use the options under the Edit pull-down list.

4.5.3 Printing Data Files

You can print both graphic and tabulated data from a run.

To choose the print options, select File > Print and select the desired options in the Print dialog. For a complete description of all printing options, refer to the Experion software Help ﬁle (search term “printing data”).

To print ﬁles automatically after a run is completed:

1. Select Tools > Options and click the Advanced tab.

2. Select Auto Print and then click Settings.

3. Choose the desired options in the Experion Print dialog and click Apply.

To save or print the data as a PDF ﬁle, select File > Print and select Print PDF

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 29

Experion Automated Electrophoresis System

30 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Assessing RNA Quantity and Quality

Experion Automated Electrophoresis System

5.1 RNA Quantitation

Experion™ software begins RNA quantitation by comparing the time-corrected area (corrected area) under the electropherogram of a sample (excluding the lower marker) with the corrected area under the electropherogram of the RNA ladder. It then uses this value, the ladder concentration (160 ng/μl for the RNA StdSens assay and 1,000 pg/μl for the RNA HighSens assay), and a correction factor to determine the RNA concentration of the sample. Results are displayed under the Results and Run Summary tabs (Figure 5.1).

5.2 RNA Quality Indicator (RQI)

The RNA quality indicator (RQI) feature of Experion software allows you to estimate the level of degradation in a eukaryotic total RNA sample. The RQI algorithm compares the electropherogram of RNA samples to data from a series of standardized, degraded RNA samples and automatically returns a number between 10 (intact RNA) and 1 (degraded RNA) for each sample (Table 5.1). RQI analysis is performed for all samples with an Experion StdSens or HighSens analysis kit.

To view RQI data, select either the Results (selected sample) or Run Summary (all samples) tab (Figure 5.1).

Fig. 5.1. Exper ion quantitation results. RQI results appear with quantitation results under the Results (top) and Results Summary (bottom) tabs. E xamples shown are for a total RNA Experion RNA HighSens analysis.

32 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

Table 5.1. RQI values, indicators, and their interpretation. These values can be customized.

RQI Value Color Indicator Meaning

7–10 Green Acceptable quality

4–6 Yellow Possibly acceptable quality

1–3 Red Unacceptable quality

Experion software also issues RQI alerts when RQI calculations may not be reliable. When one of the following limitations or anomalies is detected, an alert is displayed in the Run Summary tab:

RNA conc. too low — concentration of RNA is too low (<10 ng for the RNA StdSens assay

and <200 pg for HighSens assay)

Critical anomaly — RQI calculations cannot be performed, sample should be assayed

again (if this error occurs in >1% of samples, contact Bio-Rad Technical Support)

To ensure the best results from RQI analysis, make sure that:

Ladder peaks are identiﬁed correctly

Lower marker is identiﬁed correctly

18S and 28S fragments are within normal time windows (this may require that you edit a

fragment)

For more information about how the RQI is calculated and its comparison to the RNA integrity number (RIN), see bulletin 5761.

For information about RNA degradation and its effect on data quality, see bulletins 5893, 5452, and

5315.

RQI color indicators and value assignments can be customized. The default values are approximations. Acceptable RNA quality varies depending on the application. Validate your RQI values for your application and set the ranges accordingly.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 33

Experion Automated Electrophoresis System

34 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Changing Analysis

Settings and Parameters

Once an Experion™ analysis is complete and you have examined the quality of the run and of the data, you can reanalyze the data by changing some of the settings. Most of the steps outlined in this chapter are used for troubleshooting purposes or are optional; they are not required in most analyses.

Experion Automated Electrophoresis System

6.1 Manually Setting a Marker

To designate a peak as a marker (and change the alignment of a sample), right-click on the peak in either the electropherogram (single-well view) or the Peaks tab and select Manually Set Lower Marker to deﬁne the peak as the new lower marker. Do this if no or incorrect peaks have been selected as the lower marker.

Pink triangles in the virtual gel indicate markers. The markers should be aligned across all lanes.

6.2 Changing Peak Finding Parameters

Peak ﬁnd parameters tell the peak-ﬁnd algorithm whether a peak is signiﬁcant.

To edit the parameters used by Experion software for ﬁnding peaks in a selected sample/well:

1. Select the sample by clicking on the lane in the virtual gel (or on the sample name in the tree view).

2. Under the Settings tab, edit the parameters under Peak Find Settings:

Min. Peak Height — minimum height required for a peak to be integrated (accepted values

are 0.1–1,000). Determine the appropriate value for this parameter by zooming in on a small peak in an electropherogram and reading its ﬂuorescence value on the y-axis

Min. Peak Width — minimum amount of time that must elapse before a peak is

recognized (accepted values are 0.1–10, with lower values yielding more peaks)

Slope Threshold — represents the variation in ﬂuorescence units over time required to

detect a peak (accepted values are 0.01–10, with lower values yielding more peaks)

Fig. 6.1. Peak Find S ettings options.

3. Click Apply to apply the changes and reanalyze the data. Click Save in the main screen toolbar to save the new conditions or click Reset to Default and Apply to recover the default settings.

To modify the peak-ﬁnd settings for all sample wells:

1. Under the Settings tab, click All Well Settings or select Analysis > All Wells Settings.

2. In the All Wells Settings dialog, expand Peak Find Settings and select the type of modiﬁcation you would like to apply (see above for descriptions).

3. Click OK to apply changes and Ye s to overwrite the settings. Click Save in the main screen toolbar to save the new conditions. To reset to default settings, click Reset to Default.

Do not adjust the Peak Find Settings values so that the markers are eliminated from analysis.

36 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

6.3 Changing General Settings

These settings appear in the Settings tab, in the All Wells Settings dialog under General Settings (Figure 6.2). They apply to all samples in a run.

Data Frequency — displays the rate of data collection in Hz; cannot be edited

Filter Width — determines the width of the polynomial (in sec) to be applied to the data for

ﬁltering (noise reduction). Set this variable to less than twice the width of peaks (0.3–10)

Polynomial Order — deﬁnes the power series applied to ﬁt the raw data. Valid values are

1–10, with lower values yielding smoother curves

Fig. 6.2. General Settings options.

6.4 Baseline Modification

Experion software subtracts the average baseline value from the signal. This does not affect the shape of the curve; it shifts the baseline down to zero. The default settings for baseline modiﬁcation are appropriate for most samples.

To shut off this baseline modiﬁcation method for a selected sample:

1. Under the Settings tab, deselect the Zero Baseline option under Baseline Correction.

2. Click Apply to apply the changes and reanalyze the data, and click Save in the main screen toolbar to save the new conditions or choose Reset to Default and Apply to recover the default settings.

To edit the parameters used by Experion software for adjusting the baseline for all samples:

1. Either open the Settings tab or select Analysis > All Wells Settings.

2. In the All Well Settings dialog, expand Algorithm Inclusion Settings and deselect the Zero Baseline modiﬁcation. Click OK.

3. In the Experion Application dialog, click Yes to save changes and reanalyze the data. Click Save in the main screen toolbar to save the new conditions.

To reset to default settings, click Reset to Default in the Settings tab under All Well Settings and then click Save Run in the main screen toolbar.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 37

Experion Automated Electrophoresis System

6.5 Turning Analysis Off

To turn analysis off, select Analysis > Turn Analysis Off. Analysis remains off for all runs until it is turned back on.

Turning analysis off displays the raw data from a run; it deactivates alignment, shifting the positions of the lower marker in each sample. In addition, the pink arrows (in the virtual gel) indicating the positions of the markers appear only in the ladder lane. With analysis off, the migration of the markers may not be perfectly aligned (Figure 6.3). Turning analysis off can help reveal if a problem is isolated (one or a few wells have the marker out of alignment) or is general (alignment problems across the entire chip).

Analy sis on Analy sis off

Fig. 6.3. Effects of turning analysis off.

38 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

6.6 Manual Peak Integration

This function enables deﬁnition or reﬁnement of ladder peak areas.

1. Select a sample and click or select Analysis > Manual Integration to start manual integration.

2. Adjust the baseline by clicking and dragging the dots at the ends of the peaks (Figure 6.4). If a peak in the electropherogram is not detected by the software, right-click on that peak and select Add Peak. Zoom in on the peak for a better view of the baseline.

3. Save any changes by selecting File > Save Run As (use a new ﬁle name), or revert to the original baseline and integration by clicking .

Manual integration applies to the ladder well (L). Moving back to automated integration removes changes made during manual integration unless those changes are saved. The baselines of sample rRNA peaks can be modiﬁed by repositioning the end points of the peak baseline, but this affects only rRNA ratios; concentrations will not be affected.

Fig. 6.4. Manual integration mode indicates the beginning and end of peak baselines with blue dots.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 39

Experion Automated Electrophoresis System

40 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Troubleshooting

Experion Automated Electrophoresis System

7.1 Electrophoresis and Priming Stations

Error Probable Cause Recommended Action

IV Check Failure

™

(Experion

Check Seal

(Priming station)

software)

One or more wells are not ﬁlled correctly, and the electrodes are not immersed

Air bubbles are interfering with the electrical contact in one or more of the wells

An electrode is loose or disconnected Perform a conductivity test with the cleaning chip or

Lid is not closed Close the lid

Chip is not primed properly Repeat the analysis using a new chip

Priming seal is worn, torn, or clogged Replace the priming seal (catalog #700-7031)

Chip is not positioned correctly in the priming station

Gel-stain solution (GS) was added to the wrong well for priming

Fill all sample wells of the chip with blank or replicate samples

Ensure pipets are properly calibrated

Stop the run, remove the chip, and use a clean pipet tip to remove the sample and apply a new aliquot

When pipetting, insert the tip ver tically and to the bot tom of the well. Dispense the liquid slowly. Do not expel air at the end of the pipetting step. Dispense only to the ﬁrst stop on the pipet. Positive (reverse) pipetting may also be used

conductivity chip from the Experion validation kit (catalog #700-7051). Add 850 µl water to the chip, place the chip in the electrophoresis station, and close the lid. In Experion software, select Tools > Diagnose Instrument and select Conductivity in the Instrument Diagnostics dialog. If the test passes, focus on the sources of error above. If it fails, contact Bio-Rad Technical Support with the results

Align the arrow on the chip with the arrow on the platform

The priming well is highlighted on the chip. See Section 3.5, Prime the Chip

No chip detected Chip not positioned properly, empty wells,

Run aborts before all wells are analyzed

or incor rect reagents

Computer power save r options are causing runs to abort (this is a problem only with Experion sof tware prior to version 3.0)

Make sure chip is positioned properly and correct reagents used

Upgrade to the newest software version

Conﬁrm all power saver and/or power options are turned off before starting the assay. Do not let the computer “sleep” during a run. If the computer goes to sleep, the run will abort

7.2 Experion RNA Analysis

Error Probable Cause Recommended Action

Concentrations are not reproducible (CV > 20%)

Peaks were not detected in the RNA ladder

Sample preparation procedures are inconsistent

An air bubble is at the bottom of the ladder well or microchannels due to poor priming or sample loading; air bubbles can interfere with the contact between the ele ctrode and the liquid

Use the same reagents and follow the same procedures for each sample and each run. Do not modify the protocol outlined in Chapter 3 to compensate for RNA concentration or sample variability

Check the chip for air bubbles after priming and sample loading:

After priming: turn the chip over and make sure the

microchannels are ﬁlled with gel-stain solution (GS). If the chip is primed correctly, the wells should be hard to see. Compare the primed chip to an unprimed chip if necessary. Do not touch the glass

After sample loading: tilt the chip and view it from

different angles against a light background

42 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

Error Probable Cause Recommended Action

No peaks detected in some samples

No peaks detected in any samples

There is not enough sample in the wells Ensure pipets are calibrated and that correct volumes have

Samples were not prepared properly Review the sample preparation procedure detailed in

RNA stain was photobleached Protect RNA stain and gel-stain solution (GS) from light

Particulates are clogging the microchannels Use only high-quality 0.2 µm-ﬁltered water (such as

Gel-stain solution (GS) is old or photobleached Prepare fresh GS

Chip was primed with incorrect pressure and time settings

Particulates are clogging microchannels Use only high-quality, 0.2 µm-ﬁltered water (such as

been added to the wells

Chapter 3

Prepare new GS; use a new tube of RNA stain if necessar y

Experion DEPC-treated water, not autoclaved water)

Verify that gel (G) and GS were properly ﬁltered

If using samples that contain particulates, perform a quick microcentrifuge spin of the prepared sample to pellet particulates before loading

Remove solutions from the top of the liquid to prevent introducing par ticulates pelleted during centrifugation

Check settings on the priming station and repeat analysis with a new chip

Experion DEPC-treated water, not autoclaved water)

Verify that gel (G) and GS were ﬁltered prope rly

If using samples that contain particulates, perform a quick microcentrifuge spin of the prepared sample to pellet particulates before loading

Remove solutions from top of liquid to prevent introducing particulates pelleted during centrifugation

Lower marker is missing Samples do not contain loading buf fer or were

Electropherogram peaks are much smaller than expected (peaks are <50% of what is expected)

not prepared properly

Not enough sample was added Ensure pipets are calibrated to accurately deliver ≤10 µl

Too much salt in s ample; salt ions compete with sample ions during injection

Gel-stain solution (GS) is old or photobleached Prepare fresh GS

Reagents, speciﬁcally RNA stain, were not brought to room temperature prior to use; if stain is not equilibrated to room temperature, it will be more concentrated when it is added to gel, which may result in lower peaks and areas

Filtered gel (G) or gel-stain solution (GS) were mislabeled or used improperly. RNA chips contain wells for GS that carr y sample through microchannels and supply dye for analysis

Review the sample preparation procedure in Chapter 3

Ensure RNA is in buf fer of low ionic strength (for RNA StdSens use TE or water; for RNA HighSens use water)

Analy ze a dilution series of sample in water on another chip. If peak heights and concentrations of diluted sample increase, salt is too high in the original sample. Determine proper dilution to minimize salt effect

Repeat analysis with a new chip, new tube of stain, and properly equilibrated reagents

Ensure chip was primed with GS

Ensure G was applied to well labe led G and GS was used in all wells marked GS

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 43

Experion Automated Electrophoresis System

Error Probable Cause Recommended Action

Peaks are small, broad, or missing in some samples but present in other samples on the same chip

Late migration (peaks broaden and are delayed over the course of the run)

Peaks migrating faster than usual; electropherogram appears compressed

Contaminants are present Prepare samples in a dif ferent buffer or dilute in water

Do not use autoclaved water for diluting samples or ladder. Use only high-quality 0.2 µm-ﬁltered water (such as Experion DEPC-treated water)

RNA ladder and samples were not denatured completely

Air bubbles are interfering with electrical contact in one or more of the wells

Electrophoresis station temperature is inappropriate or ﬂuctuating during run (should be 30–35°C); there is no cooling unit in the electrophoresis station, so if the temperature changes during the run, samples exhibit different separation characteristics

Heat samples and RNA ladder for 2 min at 70°C before loading

Do not use RNA ladder that has been repeatedly heated and frozen

Turn analysis off (see Section 6.5, Turning Analysis Off, for more information). Late migration is indicated if there is a drift in the migration of lower marker across the chip. Refer to the troubleshooting tips that follow

Stop run, remove chip, and use a clean pipet tip to remove or dislodge the bubbles, or remove and replace the solution in affected wells

When pipetting, insert the tip ver tically and to bottom of the well. Dispense liquids slowly. Do not expel air at the end of the pipetting step. Dispense only to the ﬁrst stop

Ensure the temperature of the room is appropriate and stable, and place the electrophoresis station away from all heat sources, such as windows or ovens

Sizing is incorrect RNA ladder peaks and lower markers were

Quantitation is incorrect Lower marker was not properly assigne d by

Ghost peaks or contaminants appear in electropherograms

improperly assigned by the software

the software

Pipetting and/or dilution errors occurred during sample preparation or chip loading

Samples are old or have not been stored properly and RNA degraded

Electrodes are contaminated Perform the deep cleaning procedure described in

Manually assign lower marker (follow instructions in Section

6.1, Manually Setting a Marker), if necessary

Check that lower marker was properly assigned. Manually select the marker, if necessary (follow instructions in Section

6.1, Manually Setting a Marker)

Ensure calculations and dilutions are correct

Ensure pipets are calibrated, and use pipets that accurately deliver volumes of ≤10 µl

Do not modify the sample preparation and loading protocols described in this manual

Ensure the total RNA concentration of samples is within the linear dynamic range; if total RNA concentration is unknown, analyze a sample dilution series

Review the essential practices described in Chapter 2 before initiating another analysis with a new chip

Prepare fresh samples and try the analysis again

Appendix B and software Help section (search term “electrodes”)

Use only 0.2 µm-ﬁltered water (such as Experion DEPCtreated water) for diluting samples and the RNA ladder; do not use autoclaved water

RNA ladder degraded Aliquot the RNA ladder for single use and store at –70°C

44 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

Error Probable Cause Recommended Action

Lower marker is identiﬁed incorrectly

Virtual gel does not resemble an agarose gel separation of same samples

Baseline is ﬂuctuating (for example, into very negative and positive values)

RNA ladder or samples appear degraded

Experion sof tware selected incorrect peak to correspond to the marker; such an error affects alignment and sizing

Gel view is a direct interpretation of electropherogram data. By default, ﬂuorescence intensity of bands in each lane is scaled to intensit y/peak height of highest peak in that sample (scale to local). Consequently, each gel lane may have a different scale of ﬂuorescence intensity

Air bubbles are interfering with electrical contact in one or more of the wells

Sample is in inappropriate sample buffer Ensure RNA is in buffer of low ionic strength (for Experion

Chip is not completely prime d Prime and load a new chip

Samples are too concentrated; RNA levels are out of range of assay

RNase contamination of electrodes, reagents, or consumables

Manually select the markers by following the instructions in Section 6.1

Adjust scaling of the virtual gel to Scale To Global (se e Section 4.2). This adjusts the scale on all electropherograms

Adjust the intensity of the vir tual gel using the sliding cursor

Stop the run, remove the chip, and remove and replace solution in affe cted wells

RNA StdSe ns use TE or water, for Exper ion RNA HighSens use water)

Dilute the samples and repeat the analysis

Perform the deep cleaning procedure described in Appendix B and software Help section (search term “electrodes”)

Use only 0.2 µm-ﬁltered water (such as Experion DEPCtreated water) for diluting samples and the RNA ladder

Use RNase-free consumables and RNase-free, ﬁlter pipet

Improper storage

tips; change pipet tips with each use

Store RNA ladder and sample s appropriately (for example, at –70°C)

7.3 Contacting Technical Support

If you are having problems with a run and would like the Bio-Rad Technical Support team to review your data, please submit the following ﬁles to LSG_TechServ_US@bio-rad.com for analysis:

Run ﬁle (C:\Program Files\Bio-Rad Laboratories\Experion Software\Data\<daily folder

name>\<run name>.bdf)

Run log (C:\Program Files\Bio-Rad Laboratories\Experion Software\Data\<daily folder

name>\<run name>.log)

Packet ﬁle (C:\Program Files\Bio-Rad Laboratories\Experion Software\Data\<daily folder

name>\Packet\<run name>.txt)

System log (C:\Program Files\Bio-Rad Laboratories\Experion Software\Backup\Version X\

Experion_System.log or .txt)

Lot numbers of the RNA chips and the Experion RNA analysis kit

To export the run ﬁle, log ﬁle, and packet ﬁle, select the run(s) in the tree view and click Export Selected Runs. The software exports the ﬁles into folders with the corresponding project name(s).

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 45

Experion Automated Electrophoresis System

46 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Appendices

Experion Automated Electrophoresis System

Appendix A: How the Experion™ System Works

The Experion automated electrophoresis system performs electrophoresis of samples within a microﬂuidic chip. Within each chip, a series of microchannels connects the sample wells to a separation channel and buffer wells. A set of electrodes in the electrophoresis station applies a voltage across the microchannels, causing charged molecules in the samples to migrate into and through the separation channel. Samples are run sequentially, with a sufﬁcient lag between them to prevent crosscontamination. For separation, the microchannels are ﬁlled with a proprietary gel-stain solution (GS) that acts as a sieving matrix; therefore, the sample RNA fragments migrate through the separation channel at a rate based on their size and charge1. Finally, the fragments interact with a ﬂuorescent dye during separation and are detected as they pass a laser and photodiode detector (laser-induced ﬂuorescence).

RNA analysis is accomplished with the Experion RNA StdSens and HighSens analysis kits, which perform analyses based on the following sample types:

mRNA

Total RNA — eukaryotic

Total RNA — prokaryotic

Overall, RNA analysis with the Experion system involves the following steps:

Preparing the chip (priming and loading) — Priming ﬁlls the microchannels of the

microﬂuidic chip with GS, which contains both the sieving matrix and ﬂuorescent dye. RNA samples and loading buffer are then added

Running the chip — The chip is inserted into the electrophoresis station, and as the

instrument lid is closed, electrodes come into contact with the solution in the wells. Voltage is applied to the sample wells of the chip, causing the charged RNA fragments to migrate into the separation channel. In the separation channel, the different fragments separate as they move at different rates through the gel matrix, depending on their size. During separation, the ﬂuorescent dye intercalates between the bases of the fragments

Detecting the fragments — As the fragments migrate toward the end of the separation

channel, a laser excites the dye, causing it to ﬂuoresce if it is bound to RNA. A photodiode detects the ﬂuorescence, and Experion software plots the ﬂuorescence intensity vs. time to produce an electropherogram and a virtual gel image

Analyzing the data — Following separation, Experion software subtracts background noise,

removes spikes, identiﬁes and integrates peaks, and assigns their sizes, concentrations, and quality (RNA quality indicator, RQI, value for eukaryotic total RNA). Following analysis, parameters may be changed and the data reanalyzed

How Experion Software Analyzes RNA Fragments

Data Presentation

As the photodiode detects ﬂuorescent signals from the dye-RNA complexes, Experion software converts the signal into an electropherogram (a plot of ﬂuorescence vs. time). Experion electropherograms generally have the following features (Figure A.1):

Sample peaks — signals generated by the sample fragments

Lower marker — signal generated by the lower marker, which is included in the RNA

loading buffer to normalize the separation of fragments across all wells in a chip

RNA samples are heat-denatured to minimize the effects of shape or structure on migration.

48 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

The appearance of the electropherogram depends on the type of RNA assay performed:

mRNA analyses often yield broad peaks or a smear and, on occasion, contaminating 18S

and 28S rRNA peaks (Figure A.1). In addition, mRNA samples require more time for analysis than total RNA samples to ensure inclusion of the population of longer transcripts and to achieve a smooth baseline

Total RNA analyses yield 2–3 prominent peaks corresponding to rRNA (18S and 28S and

occasionally 5S in eukaryotic sources, 16S and 23S in prokaryotic sources, Figure A.2)

Fig. A.1. Electropherogra m gener ated by an E xperion RNA HighSens analysis of mRNA. The 18S rRNA peak is identiﬁed by Experion sof tware.

Fig. A.2. Ele ctropherogram generated by an Experion RNA HighSens analysis of eukar yotic tot al RNA . The 18S and 28S rRNA peaks are identiﬁed by E xperion software.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 49

Experion Automated Electrophoresis System

Once separation occurs, the data are converted into a densitometric, gel-like image, or virtual gel (Figure A.3). Each lane in the virtual gel corresponds to a different sample. The sample peaks and lower marker seen in the electropherogram also appear in the virtual gel.

Lower marker

Fig. A.3. Vir tual gel generated by Experion RNA StdSens analysis of mRNA (left) a nd eukaryotic total RN A (right). Shown are separations of the RNA ladder (L) and 12 sample s on a single chip. Pink triangles mark the position of the lower marker.

Once separation is complete, the software subtracts background noise, removes spikes, identiﬁes and integrates peaks, and assigns their sizes and concentrations. The results of data analysis are tabulated and presented in a Results table at the end of analysis (Figures A.4 and A.5). The types of data available include the following:

Concentration of mRNA or total RNA (in ng/μl or pg/μl, depending on the assay)

rRNA contamination (mRNA assays only)

28S/18S ratio (eukaryotic total RNA assays) or 23S/16S ratio (prokaryotic total RNA assays)

RNA quality indicator (RQI, eukaryotic total RNA assays)

50 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Fig. A.4. Results tab generated by Experion RNA StdSens analysis of eukar yotic total RNA. Shown is the

Results tab for a single sample.

Experion RNA StdSens and HighSens Analysis Kits

Data Analysis: Normalization, Sizing, Quantitation, and Quality Determination (RQI)

Following separation, Experion software analyzes RNA fragments using one or more of the following:

Internal 50 bp lower marker for normalizing the migration times of samples in different wells

RNA ladder for determining fragment size (sizing) and concentration (quantitation)

RNA quality indicator (RQI, eukaryotic total RNA analysis only) to quantitate the state of

degradation of a sample

Normalization: Aligning Peak Migration Times

To compensate for small variations in each separation, Experion RNA analysis uses an internal 50 bp marker to normalize the migration times among samples. The marker is included in the Experion RNA loading buffer and so is added to each sample and the RNA ladder (Figures A.1–A.3). Inclusion of the marker and the normalization process ensures that the system software properly identiﬁes peaks.

RNA Sizing: Determining Peak Sizes

The ﬁrst sample to be analyzed is the RNA ladder, which contains 8 RNA fragments of 200–6,000 nt. Experion software constructs a standard curve of migration time as a function of fragment size from the RNA ladder separation. It then calculates the size of the RNA fragments from the sample wells by comparing their migration times to the standard curve.

Quantitation: Determining RNA Amount

Experion software begins RNA quantitation by comparing the time-corrected area (corrected area) under the electropherogram of a sample (excluding the lower marker) with the corrected area under the electropherogram of the RNA ladder. It then uses the corrected area, the known ladder concentration (160 ng/μl for the Experion RNA StdSens assay and 1,000 pg/μl for the Experion RNA HighSens assay), and a correction factor to determine the RNA concentration of the sample. Results are displayed under the Results and Run Summary tabs (Figures A.4 and A.5).

Quality Determination (RQI)

The RQI algorithm compares the electropherogram of RNA samples to data from a series of standardized, degraded RNA samples and automatically returns a number between 10 (intact RNA) and 1 (degraded RNA) for each sample (Figures A.4 and A.5 and Table 5.1). RQI analysis is performed for samples with an Experion StdSens or HighSens analysis kit. The RQI feature of Experion software allows you to quantitate the level of degradation in a eukaryotic total RNA sample. Refer to bulletin 5761 for more details about RQI.

Fig. A.5. Run Summar y tab. RQI results a ppear with quantitation re sults under the Results (Figure A.4) and Results Summar y tabs. E xample shown is for a total RNA Experion RNA HighSens analysis.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 51

Experion Automated Electrophoresis System

How Experion Analysis Differs from Gel-Based Methods

Though the Experion RNA analysis kits and other gel-based electrophoresis methods both use a gel matrix and voltage differences to separate RNA fragments according to their charge:mass ratio, there are a number of fundamental differences between the two methods that may affect how speciﬁc fragments are separated and how data are interpreted (Table A.1).

Table A.1. Comparison of Experion RNA analysis and agarose gel electrophoresis.

Aspect Agarose Gel Electrophoresis Experion RNA Analysis

Sieving polymer (matrix) Formulation can be changed to affect resolution Fixed formulation

Agarose Polymer (not crosslinked)

RNA stain Multiple available Single, proprietary stain

Intercalates RNA fragments Intercalates RNA fragments

Detection Separate staining step; fragments stained after

separation

Separate destaining step Destaining not required

Requires photography or other imaging equipment for documentation, analysis

Types of separation Native and partially to completely denaturing Partially denaturing

Downstream applications Separated fragments available for excision,

electroelution, blotting applications

Reagent and sample usage Moderate: ng to µg sample, ml to L reagents Minimal: pg to µg sample, µl reagents

Waste generation Moderate to extensive (ml to L) Minimal (µl)

Overall analysis time Hours (depends on gel size, staining method,

and methods of documentation and analysis)

Data analysis Manual; requires additional equipment and

software

Integrated separation step; fragments stained during separation

Laser excites the stain, detection using a photodiode, both incorporated

Fragments not available for further analysis

30 min/chip (11 or 12 samples per chip)

Automated (with ﬂexibility of manual adjustments)

52 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

Appendix B: Deep Cleaning Procedure

This manual procedure applies Experion™ electrode cleaner directly to the 16 platinum pins in the electrode manifold. It is the most stringent method of cleaning the Experion electrode pins and helps ensure RNase-free operation of the system.

The need for this deep cleaning will vary from lab to lab and depends on the frequency of use of the Experion system and on the types of assays (protein, RNA, or DNA analysis) that are performed.

Perform deep cleaning:

Prior to ﬁrst use of the Experion electrophoresis station

Between a protein and DNA or RNA analysis (an Experion electrophoresis station used to

analyze protein samples will become contaminated with RNases and should be cleaned prior to any RNA analysis), or between a DNA and RNA analysis

Whenever RNase contamination is suspected or other contamination (for example, salt

deposits or other precipitates) is visible on the electrodes

Whenever a chip has been left in the electrophoresis station for an extended period of time

(for example, overnight)

Perform this procedure with the electrode manifold in place (installed in the electrophoresis station). It requires the following supplies:

Experion electrode cleaner (catalog #700-7252)

Experion DEPC-treated water (catalog #700-7253)

Foam cleaning swabs (catalog #700-7264)

Warning: Keep the chip platform dry during this procedure. Either cover the chip platform using plastic wrap and a paper towel or foil, or place a cleaning chip on the chip platform.

1. Shut off power to the electrophoresis station.

2. Add 0.5–1 ml Experion electrode cleaner to a microcentrifuge tube. Insert a swab into the solution until it is saturated with the electrode cleaner.

3. Use the swab to gently scrub each electrode pin one at a time, on all four sides. Do not press too hard on the pins, as they may bend. Finally, clean the tip of each pin. Add more electrode cleaner to the swab as necessary.

-Or-

Move the swab up and down, and side to side, 2–3 times along the columns and rows of pins.

Finally, clean the tip of each of pin. Add more electrode cleaner to the swab as necessary.

4. Repeat steps 2 and 3 twice, each time with a fresh swab soaked in DEPC-treated water.

5. Fill a cleaning chip with 800 μl DEPC-treated water, place it in the electrophoresis station, and close the lid for 5 min.

6. Remove the chip and allow the pins to dry completely by leaving the lid open for 5–10 min.

Refer to the Experion system manual for more details and images.

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 53

Experion Automated Electrophoresis System

Appendix C: Glossary

All-wells view Mode of display within the electropherogram view wherein electropherograms of all wells are shown

Baseline

Line of initial data that serves as the control, or signal representing no ﬂuorescence

Chip

Microﬂuidic device used to perform separation using the Experion system

Electropherogram

Trace recording of signal (ﬂuorescence intensity) as a function of time; displayed in the electropherogram view

Electropherogram view Portion of the Experion software user interface dedicated to display of electropherogram data

Electrophoresis station Instrument that houses all the electrical, optical, and hardware components necessary to perform separation, staining, destaining, band detection, and imaging of samples; equipped with a universal power supply to operate at 100–240 V, 50–60 Hz

Gel priming well Well of the microﬂuidics chip used to ﬁll the microchannels of the chip with gel-stain solution (GS) during priming

Gel-stain solution (GS)

A mixture of gel matrix and stain; used for sample separation and detection

Gel view

Portion of the Experion software user interface dedicated to display of virtual gel data

LabChip

Microﬂuidic “lab-on-a-chip” technology developed by Caliper Life Sciences; the Experion automated electrophoresis system was built on LabChip technology

Lower marker

RNA fragment (50 bp) included in the Experion RNA sample buffer and used to normalize the separation of fragments across all wells in the chip

Microchannels Fine channels approximately the diameter of a human hair are used for samples separation in Experion microﬂuidic chips

Microﬂuidic technology (microﬂuidics)

Multidisciplinary ﬁeld comprising physics, chemistry, engineering, and biotechnology that deals with the behavior, precise control, and manipulation of microliter and nanoliter volumes

Normalization

Alignment of peak migration times across different sample wells of the same chip; the lower marker is used by Experion software for this purpose

Overlay

Function of Experion software that allows different electropherograms to be superimposed for easier comparison

Peak

Signal in an electropherogram generated and identiﬁed by the peak-ﬁnd settings to be signiﬁcantly different from the baseline

Priming

Filling the microchannels of the microﬂuidic chip with gel-stain solution in preparation for analysis

Priming station Instrument used to prepare chips for sample analysis; applies pressure to the priming well of the microﬂuidic chip and ﬁlls the network of microchannels with the gel-stain solution

Project folder

User-deﬁned folder containing multiple run ﬁles comprising a particular project

Project tree

Hierarchical tree of all saved projects and the data ﬁles they contain

Quantitation

Determination of the concentration of an RNA sample

Results and settings Portion of the Experion software user interface dedicated to display of tabulated analysis results and analysis settings and parameters

54 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

RNA ladder

RNA standard containing 8 RNA fragments of 200–6,000 nt; separation data from the ladder are used by Experion software for size determination of sample fragments and quantitation of RNA samples

RNA quality indicator (RQI) Quantitative measure of integrity of eukaryotic total RNA, generated by Experion software and ranging from 1 (completely degraded) to 10 (intact)

Run ﬁle

File containing data for a single run

Single-well view

Function of Experion software that allows an electropherogram from a single well to be viewed

Sizing

Determination of the size, in nt, of an RNA fragment

Experion RNA StdSens and HighSens Analysis Kits

Spin ﬁlter

Polypropylene spin tubes with ﬁlter membranes used to remove particulate matter from the gel-stain (GS) and gel (G) by centrifugation

Tree view

Portion of the Experion software user interface dedicated to display of the project tree; used to locate and manipulate data ﬁles and organize runs into projects

Virtual gel

Densitometric, gel-like image generated from electropherogram data and displaying peaks as bands; displayed in the gel view

Vortex station

Instrument used to mix RNA or DNA samples and loading buffers (supplied in the kits) in the chip wells; used for nucleic acid applications only

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 55

Experion Automated Electrophoresis System

Appendix D: Ordering Information

Catalog # Description

700-7001 Experion™ System, 100–240 V, for RNA and DNA analysis, includes electrophoresis station, priming station, vortex station, software, USB2 cable

701-7001 Experion System, 100–240 V, for RNA and DNA analysis, includes electrophoresis station, priming station, vortex station, software, USB2 cable, Experion RNA StdSens starter kit

700-7111 Experion RNA StdSens Starter Kit, includes 3 RNA StdSens chips, 2 cleaning chips, Experion reagents, 2 spin ﬁlters, total RNA standard, cleaning swabs, electrode cleaner, tips, and tubes to run 3 chips

700-7103 Experion RNA StdSens Analysis Kit for 10 Chips, includes 10 RNA StdSens chips, 2 cleaning chips, 1,250 μl RNA gel, 20 μl RNA StdSens stain, 20 μl RNA ladder, 900 μl RNA StdSens loading buffer, 2 spin ﬁlters

700-7105 Experion RNA HighSens Analysis Kit for 10 Chips, includes 10 RNA HighSens chips, 2 cleaning chips, 1,250 μl RNA gel, 20 μl RNA HighSens stain, 20 μl RNA ladder, 900 μl RNA HighSens loading buffer, 100 μl RNA sensitivity enhancer, 2 spin ﬁlters

700-7153 Experion RNA StdSens Chips, 10, plus 2 cleaning chips

700-7154 Experion RNA StdSens Reagents and Supplies for 10 Chips, includes 1,250 μl RNA gel, 20 μl RNA StdSens stain, 20 μl RNA ladder, 900 μl RNA StdSens loading buffer, 2 spin ﬁlters

700-7155 Experion RNA HighSens Chips, 10, plus 2 cleaning chips

700-7156 Experion RNA HighSens Reagents and Supplies for 10 Chips, includes 1,250 μl RNA gel, 20 μl RNA HighSens stain, 20 μl RNA ladder, 900 μl RNA HighSens loading buffer, 100 μl RNA sensitivity enhancer, 2 spin ﬁlters

700-7251 Experion Cleaning Chips, 10

700-7254 Experion Spin Filters, 10

700-7252 Experion Electrode Cleaner, 250 ml

700-7253 Experion DEPC-Treated Water, 100 ml

700-7051 Experion Validation Kit, 3 test chips, qualiﬁcation procedures, dongle

700-7264 Cleaning Swabs, lint free, for electrode deep cleaning, 25

700-7031 Experion Priming Seals, 2

56 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 57

Experion Automated Electrophoresis System

58 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Experion RNA StdSens and HighSens Analysis Kits

Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 59

Bio-Rad Laboratories, Inc.

Life Science Group

Bulle tin 10000 976 Rev C US/EG

Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 20 8732 2339 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000

1110 Sig 1109

Bio-Rad Experion RNA Analysis Kits User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

1.1 Product Description

1.2 Kit Components

1.3 Storage Conditions

1.4 Additional Requirements

Essential Practices

2.1 Storing and Preparing Samples and Reagents

2.2 Priming and Loading the Chip

2.3 Running the Analysis

2.4 General Maintenance

2.5 Experion Video Tutorials

3.1 Set Up the Electrophoresis Station

3.2 Equilibrate the Kit Reagents

3.3 Filter the Gel and Prepare the Gel-Stain Solution

3.4 Prepare the Samples and the RNA Ladder

3.4.1 Experion RNA StdSens Analysis

3.4.2 Experion RNA HighSens Analysis

3.5 Prime the Chip

3.6 Load the Chip

3.7 Run the RNA Analysis

3.8 Clean the Electrodes

3.9 Evaluate the Run

Data Analysis

4.1 Viewing Data

4.1.1 Managing Run Files and Project Folders in the Tree View

4.1.2 General Display Controls

4.1.3 Electropherogram View

4.1.4 Gel View

4.1.5 Results and Settings

4.2 Changing the Fluorescence Intensity Scale

4.3 Using Results and Settings to View and Annotate Data

4.4 Comparing Data from Different Runs

4.5 Saving, Exporting, and Printing Data

4.5.1 Saving Data Files

4.5.2 Exporting Data Files to Other Applications

4.5.3 Printing Data Files

Assessing RNA Quantity and Quality

5.1 RNA Quantitation

5.2 RNA Quality Indicator (RQI)

6.1 Manually Setting a Marker

6.2 Changing Peak Finding Parameters

6.3 Changing General Settings

6.4 Baseline Modification

6.5 Turning Analysis Off

6.6 Manual Peak Integration

Troubleshooting

7.1 Electrophoresis and Priming Stations

7.2 Experion RNA Analysis

7.3 Contacting Technical Support

Appendices

Appendix B: Deep Cleaning Procedure

Appendix C: Glossary

Appendix D: Ordering Information