Bio-Rad Experion RNA Analysis Kits User Manual
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.
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or use of this information.
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or providing clinical information or clinical analysis, in any event in return for compensation by an unrelated party.
Copyright © 2010 Bio-Rad Laboratories, Inc.
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
1
Experion™ RNA
Analysis Kits
1
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.
4
1
2
5a
3
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),
5b
standard-sensitivit y RNA (StdSens kit),
high-sensitivity RNA (HighSens kit), and DNA
(DNA 1K and 12K kits) analyses.
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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
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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)
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2
Essential Practices
5
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):
n
Insert the pipet tip all the way to the bottom of the chip well when dispensing liquids
(this reduces the possibility of trapping air)
n
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
n
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.
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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:
n
Prior to first use of the Experion electrophoresis station
n
Whenever contamination is suspected or visible (for example, salt deposits or other
precipitates) on the electrodes
n
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
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Experion Automated Electrophoresis System
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Experion™ RNA Assay
3
Procedure
For an abbreviated version of this protocol, refer to the Quick Guide provided with the kit.
9
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:
n
A green dot and the last 4 digits of the instrument serial number appear at the lower right of the
software screen
n
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):
n
RNA samples
n
RNA ladder (red cap)
3. Equilibrate the following kit components to room temperature (15–20 min):
n
RNA stain (blue cap)
n
RNA loading buffer (yellow cap)
n
RNA gel (green cap) (if filtered gel was prepared previously, remove it from storage and
equilibrate to room temperature)
n
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.
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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.
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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.
3.
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.
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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.
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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.
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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:
n
The electropherogram of the sample being separated appears in the electropherogram
view
n
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).
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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):
n
Lower marker peak at ~20 sec, at least 5 fluorescence units above the baseline, and
distinguishable from the ladder peaks
n
Eight resolved ladder peaks
n
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:
n
Lower marker peak at ~20 sec, at least 5 fluorescence units above the baseline, and
distinguishable from the sample peaks
n
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)
n
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.
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