Thermo Fisher Scientific OpenArray Quick Start Guide

QUICK REFERENCE

Respiratory Tract Microbiota P

roﬁling Experiments v2 using

OpenArray™ plates

Pub. No. MAN0019509 Rev. A.0

Note: For safety and biohazard guidelines, see the “Safety” appendix in the Respiratory Tract Microbiota Proﬁling Experiments v2 using OpenArray™ Application Guide (Pub. No. MAN0019506). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

This document is intended as a benchtop reference for experienced users. See “Related documentation” on page 5 for resources that contain detailed instructions and troubleshooting.

Perform preampliﬁcation and dilute samples

Perform preampliﬁcation

Note: Preampliﬁcation of the TrueMark™ Respiratory Panel 2.0 Ampliﬁcation Control is not recommended.

1. Prepare PreAmp Reaction Mix: Combine the following components for the number of required reactions plus 10% overage, then mix thoroughly by pipetting up and down.

4. Gently vor

tex the plate for 10 seconds to mix, then brieﬂy

centrifuge to bring contents to the bottom of the wells.

5. Place the plate in a thermal cycler that is programmed with the following thermal cycling conditions, then start the run.

Stage Step Temperature Time

Hold

Hold Activation 95°C 2 minutes

Cycling (14

cycles)

Hold Inactivation 99.9°C 10 minutes

Hold — 4°C Hold

[1]

eat-labile UNG is completely inactivated during the initial ramp to 95°C.

UNG

incubation

Reverse

anscription

Denaturation 95°C 15 seconds

Annealing/

Extension

[1]

25°C 2 minutes

50°C 30 minutes

60°C 2 minutes

Component

TaqPath™ 1-Step RT-qPCR Master Mix, CG

TrueMark™ Respiratory Panel 2.0 PreAmp

[1]

Primers

[1]

he TrueMark™ Respiratory Panel 2.0 PreAmp Primers contains primers for all respiratory tract microbiota assays plus the TaqMan™ Universal RNA Spike In/Reverse Transcription (Xeno) Control and the RNase P RPPH1 gene, but does not contain primers for the TaqMan™ Universal Extraction Control Organism (B. atrophaeus).

Volume per

eaction

2.5 µL

2. Distribute PreAmp Reaction Mix, then Nuclease-free Water or sample nucleic acid to the appropriate wells of a 96-well plate.

No-template

Component Sample reaction

PreAmp Reaction Mix

Sample DNA or NEC

Nuclease-free Water — 5 µL

Total volume per reaction

5 µL 5 µL

5 µL —

10 µL 10 µL

contr

reaction

ol (NTC)

6. Store the plate on ice until dilution for PCR (see “Dilute the

preampliﬁed sample” on page 1).

Dilute the preampliﬁed sample

To determine dilution volumes, ﬁrst determine the total volume of diluted preampliﬁed sample that is required for PCR (see “Prepare and run respiratory tract microbiota proﬁling experiments with OpenArray™ plates” on page 2). We recommend that you prepare only the volume of diluted preampliﬁed sample that is required for your experiment. The undiluted preampliﬁed sample can be stored at –20°C long term.

1. Vortex, then brieﬂy centrifuge the plate that contains the completed preampliﬁcation sample reactions.

2. Remove the adhesive ﬁlm from the plate.

3. Prepare a 1:10 dilution of the preampliﬁed samples in a new

96-well plate.

a. Transfer the desired volume of the preampliﬁed

samples to a new 96-well plate (for example, 2 µL).

b. Add the appropriate volume of Nuclease-free Water to

each sample and control well (for example, 18 µL).

4. Seal the plate with new adhesive ﬁlm.

3. Seal the plat

e with adhesive ﬁlm.

For Research Use Only. Not for use in diagnostic procedures.

5. Vortex the plate for 10 seconds, then brieﬂy centrifuge.

b. Select the folders indicat

ed in this table:

6. Proceed directly to PCR (see “Prepare and run respiratory tract microbiota proﬁling experiments with OpenArray

™

plates” on page 2 ).

Seal the plate that contains the unused portion of the undiluted preampliﬁed samples, then store at –20°C.

Prepare and run respiratory tract microbiota proﬁling experiments with OpenArray™ plates

One-time procedures

Set up default folders and software preferences

Set up the default ﬁle locations and preferences before using the OpenArray™ AccuFill™ system for the ﬁrst time. You must be logged in as an administrator.

1. Create the following four folders in a convenient location on the same computer drive as the AccuFill™ software:

• TPF Files

• Sample Tracker 96-well Input

• Sample Tracker 384-well CSV Files

• Loaded TPF Files

2. (Optional) Copy a template ﬁle into the OpenArray™ Sample Tracker Software folder.

• Navigate to this folder on your computer: <drive>:\Program Files

(x86)\Applied Biosystems\OpenArray Sample Tracker\examples.

• Copy the 96‑Well Sample Plate 1.csv template

ﬁle, which is provided with the OpenArray™ Sample Tracker Software.

• Paste the template ﬁle into the Sample Tracker 96‑well Input folder.

AccuFill

OpenArray Plate File Input Folder

Sample Plate File Folder

Loaded OpenArray Plate File Folder

5. In the QuantS

™

folder

™

Default folder Folder contents

TPF Files TPF ﬁles for the

Sample Tracker 384-well CSV Files

Loaded TPF Files Integrated TPF ﬁles

OpenArray™ plates, with assay name and location

CSV 384-well sample plate layout

ﬁles

generated during processing with the AccuFill™ software.

tudio™ 12K Flex Software, select Tools4 Preferences4OpenArray, then select the Loaded TPF Files folder for the software Setup Folder.

Note: If the QuantStudio™ 12K Flex Software is not on the same computer as the AccuFill™ software, transfer the loaded TPF ﬁles to the computer running the QuantStudio

™

12K Flex Software.

Obtain TPF ﬁles

Go to thermoﬁsher.com/OA-plateﬁles to obtain the TPF ﬁles for the TrueMark™ Respiratory Panel 2.0, OpenArray™ Plate.

Set up the optimized folder locations and software preferences before downloading TPF ﬁles to your computer.

3. In the OpenArray™ Sample Tracker Software, select View4 Preferences, then enter the following preferences:

Field Selection

Experiment Type Gene Expression

OpenArray™ Plat

Pipettor Fixed or Adjustable tip spacing

Import Data Directory Sample Tracker 96-well Input

Export Data Directory Sample Tracker 384-well CSV Files

4. In the AccuFill™ softwar

e Select the OpenArray™ format that

will be run most often, such as

Gene Expression – 112.

e, select Instrument4Edit

Preferences, then:

a. Select Require Sample Integration.

2 Respir

atory Tract Microbiota Proﬁling Experiments v2 using OpenArray™ Quick Reference

Generate 384‑well sample plate layouts in the OpenArray™ Sample Tracker Software

Before generating 384‑well sample plate layouts, see “One-time procedures” on page 2.

1. Using a spreadsheet program, create a 96‑well sample CSV

ﬁle.

CSV template ﬁles can be found at <drive>:\Program Files (x86)\Applied Biosystems\OpenArray Sample Tracker\examples

2. Open the OpenArray™ Sample Tracker Software.

3. In the Properties screen, select Gene Expression for

Experiment Type, then select the appropriate settings for OpenArray™ Plate and Pipettor.

4. In the Samples screen, click

t your 96‑well sample CSV ﬁle that you created in

impor step 1.

5. In the Sample Mapping screen, conﬁrm that the samples for a single OpenArray™ plate are assigned to one color.

Note: If necessary, correct the OpenArray™ Plate and Pipettor settings in the Properties screen.

Import, then select and

3. F

ollowing the plate layout designated in the OpenArray Sample Tracker Software, add master mix, then DNA samples, to the wells of an OpenArray™ 384-well Sample Plate.

(Optional) Use the TrueMark™ Respiratory Panel 2.0 Ampliﬁcation Control in place of diluted preampliﬁed sample, as a positive ampliﬁcation control sample.

Component

TaqMan™ OpenArray™ RealTime PCR Master Mix

Diluted preampliﬁed sample 2.5 µL 5.5 µL

Total reaction volume 5.0 µL 11.0 µL

[1]

Volume includes 10% overage.

Volume

per well

2.5 µL 5.5 µL

Volume

per sample

4. Thoroughly mix each PCR reaction by pipetting up and

down or by using the "mix" function on a multi-channel pipette.

5. Seal the OpenArray™ 384-well Sample Plate with an aluminum foil seal, remove the foil ﬂap, then mark the edges of the ﬁlled 4 × 12 area with a pen.

6. Centrifuge the plate at 1,200 × g for 1 minute.

™

[1]

6. In the Sample Mapping screen, click the 384-Well Plate

tab, then click Export4Export *.csv.

7. Select 384-Well Plate (for AccuFill), enter a ﬁle name, then save the exported ﬁle.

Plate layouts for the 384-well sample plates are saved to individual CSV ﬁles in the Sample Tracker 384-well CSV Files folder.

Set up the PCR reactions in an OpenArray™ 384-well Sample Plate

IMPORTANT! The 4 × 12 ar

Sample Plate being ﬁlled must match the area(s) designated in the OpenArray™ Sample Tracker Software for that set of samples.

1. Remove an OpenArr aside. Allow it to come to room temperature in its unopened sleeve (~15 minutes).

The OpenArray™ plate must be completely thawed before transferring reactions to it from the OpenArray™ 384-well Sample Plate created in this section.

2. Gently swirl the contents of the TaqMan™ OpenArray™ Real- Time PCR Master Mix to thoroughly mix. Do not invert the bottle.

ea(s) of the OpenArray™ 384-well

ay™ plate from the freezer and set it

7. Score the foil along the lines that were marked before centrifuging.

Do not remove the foil from the scored area at this time.

Set up the AccuFill™ instrument and software

IMPORTANT! Do not use OpenArr

that exceed the expiration date (shown on the outer box that contains the tip trays).

1. In the OpenArr

ay™ AccuFill™ software, click Setup and Load.

The Setup Load Information window appears.

2. Conﬁgure the Loading Information pane for sample integration using the 384-well sample plate CSV ﬁle and TPF

ﬁles.

a. In the Loading Information pane (top section of the

window), ensure that the Use Sample Integration checkbox is selected.

b. Click Browse to the right of the Sample Plate ﬁeld,

then select the 384-well sample plate CSV ﬁle that you generated with the OpenArray™ Sample Tracker Software in the Sample Tracker 384-well CSV Files folder.

c. Click Browse to the right of the Plate Holder Position

of the OpenArray™ plate, then select the TPF ﬁle for the OpenArray™ plate in the TPF Files folder.

ay™ AccuFill™ System Tips

Respiratory Tract Microbiota P

3. In the Select Samples to Load pane (bottom section of the window), click the corresponding 4 × 12 area of the 384-well sample plate image, then click Next.

The Setup Deck window is displayed.

roﬁling Experiments v2 using OpenArray™ Quick Reference 3

4. In the AccuFill™ instrument, ensure that:

• Tip boxes and tips are loaded as shown in the Setup Deck window.

• The lids are removed from the tip boxes.

• The waste bin in the instrument is emptied.

5. In the Setup Deck window, conﬁrm that the deck is ready:

• Select The tips are conﬁgured as shown above.

• Select The Waste Bin is empty.

Transfer reactions to the OpenArray™ plate using the AccuFill™ instrument

1. Prepare the items needed to seal the loaded OpenArray plate .

Note: The OpenArray™ plate must be sealed promptly after being loaded with the reactions, as described here.

• Ensure that the QuantStudio™ 12K Flex OpenArray Plate Press 2.0 is ready.

• Gather and remove from their packaging the following: an OpenArray™ Lid, plug, syringe with OpenArray Immersion Fluid, and syringe tip.

• Attach the syringe tip to the syringe, carefully push some of the ﬂuid through the tip to remove air bubbles, then lay the syringe aside.

2. Load the OpenArray™ plate and the OpenArray™ 384-well Sample Plate into the AccuFill™ instrument.

• OpenArray™ plate—Remove the plate from its sleeve, then place the plate in the appropriate plate holder position in the instrument. Ensure that the barcode on the OpenArray™ plate is facing left and the serial number is facing right.

• OpenArray™ 384-well Sample Plate—Place the 384well sample plate onto the deck of the instrument, then use forceps to peel the foil from the ﬁlled area of the plate.

Seal the OpenArray™ plat

IMPORTANT! Throughout this procedure, handle the

OpenArray™ plate and the OpenArray™ Case only by the edges.

Note: The OpenArray™ Case consists of the sealed OpenArray

™

plate and the OpenArray™ Lid.

1. Place the newly loaded OpenArray™ plate in the QuantStudio™ 12K Flex OpenArray™ Plate Press 2.0.

Ensure that the barcode is facing left and the serial number is facing right.

™

2. From the OpenArray™ Lid, remove the clear protective ﬁlm from the inside of the lid and the red adhesive-protective strip from around the edge of the lid.

3. Seat the lid on the OpenArray™ plate with the lid adhesive

™

against the plate.

4. Engage the press mechanism until the green ﬂashing light

™

changes to a steady green light (approximately 20 seconds).

5. Disengage the press and remove the OpenArray™ Case.

6. While holding the case by its edges, insert the prepared

syringe tip into the port in the case, then carefully inject OpenArray™ Immersion Fluid until the case is ﬁlled.

Note:

Minimize creation of air bubbles when you dispense the

ﬂuid. Leave a small bubble at the ﬁll point to prevent ﬂuid leaks

during the instrument run.

3. Close the door of the AccuFill™ instrument.

4. In the AccuFill™ software Setup Deck window, select the

following conﬁrmations:

• The OpenArray Plate is in the Plate Holder

• Remove foil from the highlighted section of the

Sample Plate

5. Click Load.

6. As soon as the Remove OpenArray Plate window

appears, open the instrument door and remove the loaded OpenArray™ plate.

7. Proceed immediately to seal the OpenArray™ plate .

Note: For best results, seal the OpenArray™ plate within

90 seconds of completion of loading to prevent evaporation.

4 Respir

The syringe tip must be in front of the array when ﬁlling the case with immersion ﬂuid.

7. While holding the case ver

tically, remove the syringe tip,

insert the screw end of the OpenArray™ plug into the port of the case, then rotate clockwise until the black handle breaks

o.

IMPORTANT! T

o avoid leaking of immersion ﬂuid, hold the

case vertically and rotate the plug slowly.

If the plug handle breaks o pr

ematurely, use a Phillips #0

screwdriver to complete this step.

8. If needed, clean the case with a laboratory wipe that has been thoroughly sprayed with ethanol, then dry the case with a clean laboratory wipe.

atory Tract Microbiota Proﬁling Experiments v2 using OpenArray™ Quick Reference

Run the OpenArray™ plate on the QuantStudio™ 12K Flex Instrument

1. On the QuantStudio™ 12K Flex Instrument touchscreen, touch to extend the instrument tray arm.

2. Remove the clear protective ﬁlm from the outside of the OpenArray™ case (sealed plate + lid).

3. Place the OpenArray™ case on the tray arm plate adapter.

• Support the case from underneath the tray arm to prevent the case from slipping through the adapter.

• Ensure that the plate barcode and serial number are facing the front of the instrument.

3. V

iew the following ROX™ image to check for loading quality

issues:

• POST-READ_CHANNEL_4.ti

4. Check the following spotﬁnding images for leaks or other displaced sample issues.

• s02_c001_t03_p0001_m1_x2_e1_cp#_spotﬁnd.ti

• s02_c040_t03_p0001_m1_x2_e1_cp#_spotﬁnd.ti

Note: The “cp#” in the image ﬁle name refers to array positions 1 through 4 within the instrument.

5. If a problem is found, view the following pre-run spotﬁnding image to determine whether the issue existed before cycling:

• s00_c001_t01_p0001_m2_x3_e1_cp#_spotﬁnd.ti

4. Touch

5. In the Home scr

to retract the instrument tray arm.

een of the QuantStudio™ 12K Flex

Software, in the Run pane, click OpenArray.

6. In the Select Instrument pane, select your instrument.

7. Click Get Plate IDs to import the barcode of the OpenArray

plate.

Once the OpenArray™ serial number appears, the loaded TPF ﬁle corresponding to the plate should appear in the Setup File ﬁeld.

If the TPF ﬁle does not appear, click Browse, then select the correct loaded TPF ﬁle from the Loaded TPF folder.

8. (Optional) Click Browse to change the Experiment File Location.

9. (Optional) Change the software-determined Experiment File Name.

10. Click Start Run.

Note: The instrument pauses at 41 or 42 seconds prior to

the end of the run. Wait for the system to complete the run before opening the EDS ﬁle.

11. Transfer the EDS ﬁle from the instrument to an accessible location for analysis.

6. View the following FAM™ images to check for ﬂuorescent abnormalities and to conﬁrm any problem seen in the spotﬁnding images:

• STAGE2_CYCLE1_CHANNEL_1.ti

• STAGE2_CYCLE40_CHANNEL_1.ti

™

7. Note any abnormalities found, as well as all other potentially relevant information related to the setup of the run.

Customer and technical support

isit thermoﬁsher.com/support for the latest service and

support information.

• Worldwide contact telephone numbers

• Product support information – Product FAQs

– Software, patches, and updates

– Training for many applications and instruments

• Order and web support

• Product documentation – User guides, manuals, and protocols

– Certiﬁcates of Analysis

– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at www.thermoﬁsher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermoﬁsher.com/support.

Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, CA 94566 USA For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

The information in this guide is subject to change without notice. DISCLAIMER: T

PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0019509

Important Licensing Information: These pr

applicable Limited Use Label Licenses. ©2021 Thermo Fisher Scientiﬁc Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. TaqMan is a

registered trademark of Roche Molecular Systems, Inc., used under permission and license.

O THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,

Revision Date Description

A.0 5 January 2021 New document.

oducts may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all

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5 January 2021

Thermo Fisher Scientific OpenArray Quick Start Guide

Specifications and Main Features

Frequently Asked Questions

User Manual