Advanced miRNA Assays - Thermo Fisher Scientific€¦ · Perform the miR-Amp reaction ... Each...

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

TaqMan® Advanced miRNA AssaysUSER GUIDE

Fixed-content, flexible-content, and custom-configured TaqMan®

OpenArray™ Plates

for use with:TaqMan® Advanced miRNA cDNA Synthesis KitPublication Number MAN0016124

Revision B.0

Manufacturer: Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, CA 94566

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

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOURUSE OF IT.

Revision history: Pub. No. MAN0016124

Revision Date Description

B.0 8 January 2018

• Added information for flexible-content and custom-configured TaqMan® OpenArray™

Plates.

• Updated file download information for the fixed-content workflow.

• Corrected plate layout image.

• Updated TaqMan® OpenArray™ Plate plate handling.

• Updated instructions to import files to run the plate on the QuantStudio™ 12K Flexinstrument.

• Provided troubleshooting for premature break of plug handle.

A.0 13 September 2017 New document.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you acceptthe terms and conditions of all applicable Limited Use Label Licenses.Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registeredtrademark of Roche Molecular Systems, Inc., used under permission and license.

©2018 Thermo Fisher Scientific Inc. All rights reserved.

Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Overview of TaqMan® OpenArray™ Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Endogenous and exogenous controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■ CHAPTER 2 Prepare cDNA templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Guidelines for RNA input . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Guidelines for preparing cDNA templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Perform the poly(A) tailing reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Perform the adaptor ligation reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Perform the reverse transcription (RT) reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Perform the miR-Amp reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ CHAPTER 3 Prepare and run TaqMan® Advanced miRNAAssays with fixed-content OpenArray™ plates . . . . . . . . . . . . . . . . . . . . . . . . . 21

Generate OpenArray™ plate layouts in the QuantStudio™ 12K Flex Software . . . . . . . . . . . 21

Set up the real-time PCR reactions in an OpenArray™ 384-well Sample Plate . . . . . . . . . . 22

Set up the AccuFill™ instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Transfer reactions to the OpenArray™ plate (AccuFill™ instrument) . . . . . . . . . . . . . . . . . . . 25

Seal the OpenArray™ plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Run the OpenArray™ plate(s) on the QuantStudio™ 12K Flex instrument . . . . . . . . . . . . . . . 27

Check the QC images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

One-time procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Download EDT files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

TaqMan® Advanced miRNA Assays User Guide—TaqMan® OpenArray™ Plates 3

■ CHAPTER 4 Prepare and run TaqMan® Advanced miRNAAssays with flexible-content and custom-configuredOpenArray™ plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Generate 384-well sample plate layouts in the OpenArray™ Sample Tracker Software . . 31

Set up the PCR reactions in an OpenArray™ 384-well Sample Plate . . . . . . . . . . . . . . . . . . . 32

Set up the AccuFill™ instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Transfer reactions to the OpenArray™ plate (AccuFill™ instrument) . . . . . . . . . . . . . . . . . . . 34

Seal the OpenArray™ plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Run the OpenArray™ plate(s) on the QuantStudio™ 12K Flex instrument . . . . . . . . . . . . . . . 36

Check the QC images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

One-time procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Set up default folders and software preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Download TPF files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

■ CHAPTER 5 Export and review TaqMan® Advanced miRNAAssay data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Export data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Prepare exported data for analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Fields for Pivot Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Troubleshoot with cycling and imaging run images (QC images) . . . . . . . . . . . . . . . . . . . . . . 43

AccuFill™ instrument plate loading errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

OpenArray™ plate assembly and handling errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

■ APPENDIX B Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Endogenous and exogenous controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Endogenous controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Exogenous controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Overview of cDNA template preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Overview of TaqMan® Advanced miRNA Assays chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . 49TaqMan® MGB probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49About the 5' nuclease assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Best practices for PCR and RT-PCR experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Detect fluorescent contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Contents

4 TaqMan® Advanced miRNA Assays User Guide—TaqMan® OpenArray™ Plates

■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Contents


Product information

■ Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■ Overview of TaqMan® OpenArray™ Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■ Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

■ Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Product description

TaqMan® Advanced miRNA Assays are pre-formulated primer and probe sets that aredesigned for analysis of microRNA (miRNA) expression levels using theQuantStudio™ 12K Flex Real-Time PCR System and the OpenArray™ AccuFill™

System. The assays can detect and quantify the mature form of the miRNA from5–10 ng of total RNA from tissue, or 2 µL of total RNA from serum or plasma. Formore information about PCR detection with TaqMan® Advanced miRNA Assays, see page 49.

The TaqMan® Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007; soldseparately) is required for preparing the cDNA template that is used with theTaqMan® Advanced miRNA Assays. The kit enables the analysis of:

• Multiple miRNAs from a single amplified sample.• Samples that are limited in quantity, including serum, plasma, or other biologicalfluids.

The procedures in this document are for use with TaqMan® Advanced miRNA Assaysin the configurations in the following table:

Configuration Description Customizable

Fixed-content TaqMan® OpenArray™

Plates

Preplated and predefined TaqMan®

Advanced miRNA Assays that aremanufactured and stocked in advance

No

Flexible-content TaqMan®

OpenArray™ Plates

TaqMan® OpenArray™ Platesconfigured with a suggested selectionof TaqMan® Advanced miRNA Assays,categorized by specific disease,pathway, or biological process

Preselected assays can besubstituted with other predesignedassays that target more relevantTaqMan® Advanced miRNA Assays

Custom-configured TaqMan®

OpenArray™ PlatesFully customizable TaqMan®

OpenArray™ Plates

Allows the configuration of theTaqMan® OpenArray™ Plates with anypredesigned assays

1


This document describes procedures to prepare cDNA templates from miRNAfollowed by PCR amplification of the cDNA template and subsequent data analysis.

First, mature miRNAs from total RNA are modified by 1) extending the 3' end of themature transcript through poly(A) addition, then 2) lengthening the 5’ end by adaptorligation. The modified miRNAs then undergo universal reverse transcription followedby amplification to increase uniformly the amount of cDNA for all miRNAs (miR-Amp reaction). For more information about cDNA synthesis of templates forTaqMan® Advanced miRNA Assays, see page 48.

The cDNA templates are then used with TaqMan® Advanced miRNA Assays forquantification of miRNA expression levels by qPCR analysis. Predesigned TaqMan®

Advanced miRNA Assays are available for most human miRNAs in miRBase (themiRNA sequence repository). For a current list of assays, go to thermofisher.com/advancedmirna.

Note: TaqMan® Advanced miRNA Assays are for analysis of mature miRNA only.For analysis of siRNA, or other small RNAs that are fewer than 200 bases in length, goto thermofisher.com/taqmanmirna.

Chapter 1 Product informationProduct description 1


http://www.thermofisher.com/advancedmirna


http://www.thermofisher.com/taqmanmirna

Overview of TaqMan® OpenArray™ Plates

TaqMan® OpenArray™ Plates enable high-throughput screening experiments that usea small amount of sample and reagent. Each plate is the size of a microscope slide andcontains 48 subarrays. Each subarray has 64 through-holes, for a total of 3,072through-holes on the plate. Each TaqMan® OpenArray™ Plate is equivalent to eight384-well plates.

Assays are pre-loaded into the through-holes during the manufacturing process. Eachthrough-hole is 300 µm in diameter and 300 µm deep. Through-holes are treated withhydrophilic coatings and the plate surface is treated with hydrophobic coatings sothat 33 nL of reagent is retained in each through-hole by surface tension.

To run a TaqMan® OpenArray™ Plate, cDNA templates are mixed with master mix,loaded into the through-holes with the automated OpenArray™ AccuFill™ System,then cycled and imaged with the QuantStudio™ 12K Flex Real-Time PCR System.

1

3

2

4

1 Subarray2 Hydrophobic coating on plate surface3 Hydrophilic coating in through-hole4 Through-hole

Chapter 1 Product informationOverview of TaqMan® OpenArray™ Plates1


Contents and storage

Table 1 TaqMan® Advanced miRNA Assays (fixed-content TaqMan® OpenArray™ Plate)

Panel Cat. No. Amount Storage[1]

TaqMan® OpenArray™ Human Advanced miRNA Panel A32710 1 plate –25°C to –15°C

[1] See packaging for expiration date.

Fixed-content TaqMan® OpenArray™ Plates contain three replicates of each assay. Youcan add different samples to each assay (biological replicates) or you can add thesame sample to each assay (technical replicates).

Table 2 TaqMan® Advanced miRNA Assays (flexible-content and custom-configured TaqMan® OpenArray™

Plates)

Format Cat. No.Number of assays +

manufacturingcontrols[1]

Number of samples Storage[2]

TaqMan® OpenArray™

Plate Advanced miRNACustom 18

A33465 16 + 2 48triplicate

(1 sample per subarray)

–25°C to –15°C



A33466 54 + 2 48no replicates

(1 sample per subarray)




(1 sample on 2 subarrays)









[1] hsa–miR–16–5p, Assay ID: 477860_mir[2] See packaging for expiration date.

To order flexible-content TaqMan® OpenArray™ Plates, go to thermofisher.com/flexiblepanels.

Chapter 1 Product informationContents and storage 1


http://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-flexible-content-panels.html?CID=fl-we115152

http://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-flexible-content-panels.html?CID=fl-we115152

To order custom-configured TaqMan® OpenArray™ Plates, go to thermofisher.com/openarray.

Download the assay files at thermofisher.com/OA-platefiles. See “Download EDTfiles“ on page 29 or “Download TPF files“ on page 39 for download instructions.

An EDT file or a TPF file is imported into the instrument software to perform real-time PCR.

• EDT file– For fixed-content TaqMan® OpenArray™ Plates– Contains the position of each assay on the plate, the run properties, and the

thermal protocol• TPF file

– For flexible-content or custom-configured TaqMan® OpenArray™ Plates– Shows the position of each assay on the plate

• AIF– Assay Information File– Describes the TaqMan® Advanced miRNA Assays– See Understanding Your Shipment (Pub. No. MAN0017153) for detailed

information about the AIF

Note: During the download, you may be asked to enter specific order numbers, serialnumbers, or product information.

Chapter 1 Product informationContents and storage1


http://www.thermofisher.com/openarray

http://www.thermofisher.com/openarray

http://www.thermofisher.com/OA-platefiles

Required materials

Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

Table 3 Recommended RNA isolation kits

Sample type Kit Cat. No.

Tissue samples mirVana™ miRNA Isolation Kit, with phenol AM1560

mirVana™ miRNA Isolation Kit, without phenol AM1561

MagMAX™ mirVana™ Total RNA Isolation Kit A27828

mirVana™ PARIS™ RNA and Native Protein Purification Kit AM1556

Serum / Plasmasamples

Total Exosome RNA and Protein Isolation Kit 4478545

MagMAX™ mirVana™ Total RNA Isolation Kit A27828

mirVana™ PARIS™ RNA and Native Protein Purification Kit AM1556

Cell samples TaqMan® MicroRNA Cells-to-CT Kit 4391848

FFPE samples RecoverAll™ Total Nucleic Acid Isolation Kit AM1975

Table 4 TaqMan® Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007,50 reactions)

Contents Storage

10X Poly(A) Buffer

–20°C

ATP, 10 mM

Poly(A) Enzyme, 5 U/µL

5X DNA Ligase Buffer

RNA Ligase, 10 U/µL

50% PEG 8000

25X Ligation Adaptor

10X RT Enzyme Mix

5X RT Buffer

20X Universal RT Primer

dNTP Mix, 100 mM

20X miR-Amp Primer Mix

2X miR-Amp Master Mix 2–4°C

Chapter 1 Product informationRequired materials 1


http://www.thermofisher.com

http://fisherscientific.com

Table 5 Other materials and equipment required for the workflow

Item Source

Real-time PCR instrument:

QuantStudio™ 12K Flex Real-Time PCR System with theOpenArray™ AccuFill™ System

Contact your localsales office

Software

OpenArray™ Sample Tracker Software (For flexible-content andcustom-configured plates)

Included withQuantStudio™ 12K

Flex Software

ImageJ (To view quality control images) imagej.nih.gov/ig

Equipment

QuantStudio™ 12K Flex OpenArray™ Plate Press 2.0 A24945

Thermal cycler, one of the following (or equivalent):

• Veriti™ Thermal Cycler

• SimpliAmp™ Thermal Cycler

• ProFlex™ PCR System

Contact your localsales office

Centrifuge, capable of spinning sample plates at 1,000 rpm MLS

Microcentrifuge MLS

Vortex mixer MLS

(Optional) Eppendorf™ MixMate™ (shaker) Fisher Scientific™

21-379-00

Pipettes MLS

(Optional) Phillips screwdriver, #0 —

Tubes, plates, and other consumables

Plastics consumables thermofisher.com/plastics

Adhesive PCR Plate Foils AB0626

OpenArray™ AccuFill™ System Tips 4458107

QuantStudio™ 12K Flex OpenArray™ Accessories Kit

Contains items to assemble 10 OpenArray™ plates:

• 12 lids and plugs

• 12 immersion fluid syringes

• 12 carriers

4469576

OpenArray™ 384-well Sample Plates 4406947 (clear)4482221 (black)

Chapter 1 Product informationRequired materials1


https://imagej.nih.gov/ij

http://www.thermofisher.com/plastics

http://www.thermofisher.com/plastics

Item Source

Forceps MLS

Aerosol-resistant barrier pipette tips MLS

Disposable gloves MLS

Reagents

TaqMan® OpenArray™ Real-Time PCR Master Mix 4462159

Nuclease-free Water AM9930

Tris–EDTA Buffer (TE, pH 8.0) AM9849

Chapter 1 Product informationRequired materials 1


For information about using endogenous or exogenous controls with TaqMan®

Advanced miRNA Assays, see page 47.

See A technical guide to identifying miRNA normalizers using TaqMan® Advanced miRNAAssays White Paper (Pub. No. COL31302 0916) for available TaqMan® AdvancedmiRNA Assays that target miRNAs with relatively constant expression levels acrossmany different sample types (available at thermofisher.com/advancedmirna; in theDocumentation section).

Table 6 Endogenous control assay

Assay name[1] Assay ID Target Sequence

hsa-miR-16-5p 477860_mir 5′-UAGCAGCACGUAAAUAUUGGCG-3′[1] TaqMan® Advanced miRNA Assays do not detect snRNAs or snoRNAs. Do not use snRNAs and snoRNAs as

endogenous controls for these assays.

Table 7 Exogenous control assays, fixed-content TaqMan® OpenArray™ Plates (forhuman samples)

Assay Name Assay ID Target Sequence[1]

ath-miR159a[2] 478411_mir 5′-UUUGGAUUGAAGGGAGCUCUA-3′

cel-miR-39-3p[3] 478293_mir 5′-UCACCGGGUGUAAAUCAGCUUG-3′[1] Oligonucleotides for exogenous controls must be 5'-phosphorylated.[2] From A. thaliana.[3] From C. elegans.

Table 8 Exogenous control assay, flexible-content and custom-configured TaqMan®

OpenArray™ Plates (for human samples)

Assay Name Assay ID Target Sequence[1]

cel-miR-39-3p[2] 478293_mir 5′-UCACCGGGUGUAAAUCAGCUUG-3′[1] Oligonucleotides for exogenous controls must be 5'-phosphorylated.[2] From C. elegans.

Endogenous andexogenouscontrols

Chapter 1 Product informationRequired materials1



Workflow

Prepare cDNA templatesInput RNA sample

▼

Perform the poly(A) tailing reaction (page 17)

▼

Perform the adaptor ligation reaction (page 18)

▼

Perform the reverse transcription (RT) reaction (page 19)

▼

Perform the miR-Amp reaction (page 20)

▼

Prepare and run assays with fixed-contentOpenArray™ plates

Generate OpenArray™ plate layouts in theQuantStudio™ 12K Flex Software

(page 21)

Prepare and run assays with flexible-contentand custom-configured OpenArray™ plates

Generate 384-well sample plate layouts in theOpenArray™ Sample Tracker Software

(page 31)

▼ ▼

Set up the real-time PCR reactions in anOpenArray™ 384-well Sample Plate (page 22)

Set up the PCR reactions in an OpenArray™

384-well Sample Plate (page 32)

▼ ▼

Set up the AccuFill™ instrument (page 24) Set up the AccuFill™ instrument (page 33)

▼ ▼

Transfer reactions to the OpenArray™ plate(AccuFill™ instrument) (page 25)

Transfer reactions to the OpenArray™ plate(AccuFill™ instrument) (page 25)

▼ ▼

Seal the OpenArray™ plate (page 26) Seal the OpenArray™ plate (page 26)

▼ ▼

Run the OpenArray™ plate(s) on theQuantStudio™ 12K Flex instrument (page 27)

Run the OpenArray™ plate(s) on theQuantStudio™ 12K Flex instrument (page 36)

▼ ▼

Check the QC images (page 28) Check the QC images (page 28)

▼ ▼

Export and review data (page 40)

Chapter 1 Product informationWorkflow 1


Prepare cDNA templates

■ Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

■ Perform the poly(A) tailing reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

■ Perform the adaptor ligation reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

■ Perform the reverse transcription (RT) reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

■ Perform the miR-Amp reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Procedural guidelines

• Prepare samples using a total RNA isolation method that preserves small RNAs.See “Required materials“ on page 11 for recommended RNA isolation kits.

• For tissue samples: Use 5–10 ng of total RNA per reaction.

Note: Sample concentration before adding to reactions should be ≤5 ng/µL.• For blood, serum, or plasma samples: Use 2 µL of sample eluent (from the sample

isolation procedure) per reaction. If RNA can be quantified, use 5–10 ng of totalRNA per reaction.

• For optimal reverse transcription, input RNA should be:– Free of inhibitors of reverse transcription (RT) and PCR– Dissolved in PCR-compatible buffer– Free of RNase activity– Nondenatured total RNA (not applicable for double-stranded templates)

IMPORTANT! Do not denature the total RNA.

• Follow best practices when working with RNA samples (see page 51).• Calculate the number of required reactions. Scale reaction components based on

the single-reaction volumes, then include 10% overage.• If using strip tubes, change to a new strip cap after each step or incubation.• See your instrument user guide for detailed instructions about using plates,

tubes, or strip tubes.

2

Guidelines forRNA input

Guidelines forpreparing cDNAtemplates


Perform the poly(A) tailing reaction

1. Thaw samples and cDNA synthesis reagents on ice, gently vortex to thoroughlymix, then centrifuge briefly to spin down the contents and eliminate air bubbles.

Note: The TaqMan® OpenArray™ Plate must be completely thawed beforetransferring reactions to it.

IMPORTANT! The 50% PEG 8000 reagent must be at room temperature for theadaptor ligation reaction (next section).

2. In a 1.5-mL microcentrifuge tube, prepare sufficient Poly(A) Reaction Mix for therequired number of reactions according to the following table.

Component 1 Rxn 4 Rxns[1] 10 Rxns[1]

10X Poly(A) Buffer 0.5 µL 2.2 µL 5.5 µL

ATP 0.5 µL 2.2 µL 5.5 µL

Poly(A) Enzyme 0.3 µL 1.3 µL 3.3 µL

RNase-free water 1.7 µL 7.5 µL 18.7 µL

Total Poly(A) Reaction Mix volume 3.0 µL 13.2 µL 33 µL

[1] Volumes include 10% overage.

3. Vortex the Poly(A) Reaction Mix to thoroughly mix the contents, then centrifugebriefly to spin down the contents and eliminate air bubbles.

4. Add 2 µL of sample to each well of a reaction plate or each reaction tube.

Note: (Optional) Before adding the sample to the reaction plate or tube, addRNase Inhibitor to each sample to minimize the effects of RNase contamination.For detailed instructions, see the documentation provided by the RNase Inhibitormanufacturer.

5. Add 3 µL of Poly(A) Reaction Mix to each well or tube.The total volume should be 5 µL per well or tube.

6. Seal the reaction plate or tubes, then vortex briefly to thoroughly mix thecontents.

7. Centrifuge the reaction plate or tubes briefly to spin down the contents andeliminate air bubbles.

8. Place the reaction plate or tubes into a thermal cycler, then incubate using thefollowing settings and standard cycling:

Step Temperature Time

Polyadenylation 37°C 45 minutes

Stop reaction 65°C 10 minutes

Hold 4°C Hold

Proceed immediately to the adaptor ligation reaction (next section).

Chapter 2 Prepare cDNA templatesPerform the poly(A) tailing reaction 2


Perform the adaptor ligation reaction

1. In a 1.5-mL microcentrifuge tube, prepare sufficient Ligation Reaction Mix for therequired number of reactions according to the following table.


5X DNA Ligase Buffer 3 µL 13.2 µL 33 µL

50% PEG 8000[2] 4.5 µL 19.8 µL 49.5 µL

25X Ligation Adaptor 0.6 µL 2.6 µL 6.6 µL

RNA Ligase 1.5 µL 6.6 µL 16.5 µL


Total Ligation Reaction Mix volume 10 µL 44 µL 110 µL

[1] Volumes include 10% overage.[2] 50% PEG 8000 is very viscous, follow the Important statement below to ensure accurate pipetting.

IMPORTANT! For accurate pipetting of 50% PEG 8000:

· Use 50% PEG 8000 at room temperature.· Aspirate and dispense solution slowly.

a. Hold the pipette tip in the solution for ~10 seconds after slowly releasingthe plunger during aspiration. This action allows the solution to be fullydrawn into the pipette tip.

b. Keep the plunger depressed for ~10 seconds to allow the solution to befully dispensed into the Ligation Reaction Mix.

2. Vortex the Ligation Reaction Mix to thoroughly mix the contents, then centrifugebriefly to spin down the contents and eliminate air bubbles.

3. Transfer 10 µL of the Ligation Reaction Mix to each well of the reaction plate oreach reaction tube containing the poly(A) tailing reaction product.The total volume should be 15 µL per well or tube.

4. Seal the reaction plate or tubes, then vortex briefly or shake (1,900 rpm for1 minute with an Eppendorf™ MixMate™) to thoroughly mix the contents.

IMPORTANT! If vortexing, watch for a swirling motion of the adaptor ligationreaction to ensure proper mixing. Proper mixing is necessary for efficientligation.

5. Centrifuge the reaction plate or tubes briefly to spin down the contents.



Ligation 16°C 60 minutes

Hold 4°C Hold

Proceed immediately to the reverse transcription (RT) reaction (next section).

Chapter 2 Prepare cDNA templatesPerform the adaptor ligation reaction2


Perform the reverse transcription (RT) reaction

1. In a 1.5-mL microcentrifuge tube, prepare sufficient RT Reaction Mix for therequired number of reactions according to the following table.


5X RT Buffer 6 µL 26.4 µL 66 µL

dNTP Mix (25 mM each) 1.2 µL 5.3 µL 13.2 µL

20X Universal RT Primer 1.5 µL 6.6 µL 16.5 µL

10X RT Enzyme Mix 3 µL 13.2 µL 33 µL


Total RT Reaction Mix volume 15 µL 66 µL 165 µL


2. Vortex the RT Reaction Mix to thoroughly mix the contents, then centrifugebriefly to spin down the contents and eliminate air bubbles.

3. Transfer 15 µL of the RT Reaction Mix to each well of the reaction plate or eachreaction tube containing the adaptor ligation reaction product.The total volume should be 30 µL per well or tube.





Reverse transcription 42°C 15 minutes

Stop reaction 85°C 5 minutes

Hold 4°C Hold

Proceed to the miR-Amp reaction (next section) or store the RT reaction product at–20°C for up to 2 months.

Chapter 2 Prepare cDNA templatesPerform the reverse transcription (RT) reaction 2


Perform the miR-Amp reaction

1. In a 1.5-mL microcentrifuge tube, prepare sufficient miR-Amp Reaction Mix forthe required number of reactions according to the following table.


2X miR-Amp Master Mix 25 µL 110 µL 275 µL

20X miR-Amp Primer Mix 2.5 µL 11 µL 27.5 µL

RNase-free water 17.5 µL 77 µL 192.5 µL

Total miR-Amp Reaction Mix volume 45 µL 198 µL 495 µL


2. Vortex the miR-Amp Reaction Mix to thoroughly mix the contents, thencentrifuge briefly to spin down the contents and eliminate air bubbles.

3. Transfer 45 µL of the miR-Amp Reaction Mix to each well of a new reaction plateor reaction tube.

4. Add 5 µL of the RT reaction product to each reaction well or each reaction tube.The total volume should be 50 µL per well or tube.



7. Place the reaction plate or tubes into a thermal cycler, then incubate using thefollowing settings, maximum ramp speed, and standard cycling:

Step Temperature Time Cycles

Enzyme activation 95°C 5 minutes 1

Denature 95°C 3 seconds18–22

Anneal/Extend 60°C 30 seconds

Stop reaction 99°C 10 minutes 1

Hold 4°C Hold 1

Proceed to performing the real-time PCR (next section) or store the undilutedmiR-Amp reaction product at –20°C for up to 2 months.

Chapter 2 Prepare cDNA templatesPerform the miR-Amp reaction2


Prepare and run TaqMan® AdvancedmiRNA Assays with fixed-content

OpenArray™ plates

■ Generate OpenArray™ plate layouts in the QuantStudio™ 12KFlex Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

■ Set up the real-time PCR reactions in an OpenArray™ 384-wellSample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■ Set up the AccuFill™ instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

■ Transfer reactions to the OpenArray™ plate (AccuFill™ instrument) . . . . . . . . . 25

■ Seal the OpenArray™ plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

■ Run the OpenArray™ plate(s) on the QuantStudio™ 12K Flex instrument . . . . 27

■ Check the QC images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

■ One-time procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

The workflow for fixed-content OpenArray™ plates uses QuantStudio™ 12K FlexSoftware to generate the plate layouts.

Generate OpenArray™ plate layouts in the QuantStudio™ 12K FlexSoftware

For fixed-content OpenArray™ plates, use the QuantStudio™ 12K Flex Software tomap assays and samples onto the TaqMan® OpenArray™ Plates.

Download the EDT file at thermofisher.com/OA-platefiles.

See page 29 for detailed instructions.

1. In the menu, click Open, then navigate to and select the EDT file.

2. In the Home screen, in the Setup menu, click Experiment Setup.

3. Enter the Experiment Name and the Barcode.

4. In the Setup menu, Click Define, then enter the sample names in the Samplespane.The plate layout included in the EDT file is designed for one sample (threetechnical replicates) or three samples (three biological replicates) per reaction.

5. Click File4Save as to save the assay information as an EDS file.

3



Set up the real-time PCR reactions in an OpenArray™ 384-wellSample Plate

IMPORTANT! The 4 × 12 areas of the 384-well plate being filled must match the areadesignated in the QuantStudio™ 12K Flex Software for that set of samples.

1. Remove the OpenArray™ plate from the freezer; allow it to come to roomtemperature in its unopened sleeve (~15 minutes).The OpenArray™ plate must be completely thawed before transferring reactionsto it from the 384-well sample plate.

2. Prepare a 1:20 dilution of the cDNA template (the miR-Amp reaction product) in0.1X TE buffer.

3. Gently swirl the contents of the TaqMan® OpenArray™ Real-Time PCR MasterMix to thoroughly mix. Do not invert the bottle.

4. Combine master mix and cDNA samples in tubes, strip tubes, or a 96-well plate.

ComponentVolume per well

(OpenArray™ 384-well Sample Plate)

Volume per sample(16 subarrays)[1]

TaqMan® OpenArray™ Real-TimePCR Master Mix

2.5 µL 50 µL

Diluted cDNA template 2.5 µL 50 µL

Total reaction volume 5.0 µL 100 µL


5. Following the plate layout designated in the EDS file created in theQuantStudio™ 12K Flex Software, add 5.0 µL of the combined master mix andcDNA sample to each well in the sets of 4 × 4 wells in an OpenArray™ 384-wellSample Plate.

1

2

1 One full array2 One 4 × 4 set of an array (16 subarrays, 1 sample)

Chapter 3 Prepare and run TaqMan® Advanced miRNA Assays with fixed-content OpenArray™ platesSet up the real-time PCR reactions in an OpenArray™ 384-well Sample Plate3


If strip tubes or plates are used, load alternate wells to correspond to a 4 × 4 set ofthe array.

1

2

1 Samples added to alternating tubes or wells2 4 × 4 sets of an array

6. Thoroughly mix each PCR reaction by pipetting up and down or by using the"mix" function on a multi-channel pipette.

7. Seal the plate with an aluminum foil seal, remove the foil flap, then mark theedges of the filled 4 × 12 area with a pen.

8. Centrifuge the plate at 1,000 rpm for 1 minute.

9. Score the foil along the lines that were marked before centrifuging.Do not remove the foil from the scored area at this time.

Chapter 3 Prepare and run TaqMan® Advanced miRNA Assays with fixed-content OpenArray™ platesSet up the real-time PCR reactions in an OpenArray™ 384-well Sample Plate 3


Set up the AccuFill™ instrument

IMPORTANT! Do not use OpenArray™ AccuFill™ System Tips that exceed theexpiration date (shown on the outer box that contains the tip trays).

1. In the OpenArray™ AccuFill™ software, click Setup and Load.

2. In the Setup Load Information window, enter the OpenArray™ plate barcodes inthe appropriate plate holder positions.

Note: Ensure that the Use Sample Integration checkbox is deselected.

1

1 Use Sample Integration checkbox

3. Click the corresponding 4 × 12 area of the 384-well plate, then click Next to openthe Setup Deck window.

Chapter 3 Prepare and run TaqMan® Advanced miRNA Assays with fixed-content OpenArray™ platesSet up the AccuFill™ instrument3


4. Ensure that:• Tip boxes are loaded in the AccuFill™ instrument as illustrated in the

following figure:

• Lids are removed from the tip boxes.• The waste bin in the instrument is emptied.

5. In the Setup Deck window, select:• The tips are configured as shown above• The Waste Bin is empty

Transfer reactions to the OpenArray™ plate (AccuFill™ instrument)

Ensure that the OpenArray™ plate is thawed and that the entire plate is at roomtemperature.

1. Prepare the items needed to seal the OpenArray™ plate (next section).

Note: The OpenArray™ plate must be sealed promptly after being loaded withthe reactions (this section).

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

b. Gather and remove from packaging an OpenArray™ lid, plug, syringe withOpenArray™ Immersion Fluid, and syringe tip.

c. Attach the syringe tip to the syringe and carefully push some of the fluidthrough the tip to remove air bubbles, then lay the syringe aside.

2. Remove the OpenArray™ plate from its sleeve and place it in the plate holder ofthe AccuFill™ instrument.Ensure that the bar code on the OpenArray™ plate is facing left and the serialnumber is facing right.

3. Using forceps, peel the foil from the filled area of the OpenArray™ 384-wellSample Plate.

4. Close the instrument door.

Chapter 3 Prepare and run TaqMan® Advanced miRNA Assays with fixed-content OpenArray™ platesTransfer reactions to the OpenArray™ plate (AccuFill™ instrument) 3


5. In the AccuFill™ software Setup Deck window, select the followingconfirmations, then click Load.

• The OpenArray Plate is in the Plate Holder• Remove foil from the highlighted section of the Sample Plate

6. As soon as the Remove OpenArray Plate window appears, open the instrumentdoor, then remove the loaded OpenArray™ plate.

7. Proceed immediately to seal the OpenArray™ plate (next section).

Note: For best results, seal the OpenArray™ plate within 90 seconds ofcompletion of loading, to prevent evaporation.

Seal the OpenArray™ plate

IMPORTANT! Handle the OpenArray™ plate and case only by the edges throughoutthis procedure.

1. Place the filled OpenArray™ plate in the QuantStudio™ 12K Flex OpenArray™

Plate Press 2.0.Ensure that the bar code is facing left and the serial number is facing right.

2. Remove the clear plastic sheet fromthe inside of the lid, remove the redprotective film around the edge of theOpenArray™ lid, then seat the lid onthe OpenArray™ case in the platepress.

3. Engage the press mechanism until thegreen flashing light changes to asteady green light (~20 seconds).

4. Disengage the press, then remove theOpenArray™ case.

2

1

4

3

1 Protective film (remove)2 Adhesive3 Protective film (remove)4 Notched end (align with serial number)

Chapter 3 Prepare and run TaqMan® Advanced miRNA Assays with fixed-content OpenArray™ platesSeal the OpenArray™ plate3


5. While holding the OpenArray™ case by the edges, insert the prepared syringe tipinto the port in the case, then carefully inject immersion fluid until the case isfilled.

Note: Minimize creation of air bubbles when you dispense the fluid; one smallair bubble in the case is acceptable.

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

6. While holding the case vertically, remove the syringe tip, insert the screw end ofthe OpenArray™ plug into the port and rotate clockwise until the black handlebreaks off.

IMPORTANT! To avoid leaking of immersion fluid, hold the case vertically androtate the plug slowly.

If the plug handle falls of prematurely, use a Phillips #0 screwdriver to completethe step.

7. Clean the case with a laboratory wipe that has been thoroughly sprayed withethanol, then dry the case with a clean laboratory wipe.

Run the OpenArray™ plate(s) on the QuantStudio™ 12K Flexinstrument

1. On the instrument touchscreen, touch to extend the loading arm, then placethe OpenArray™ plates on the plate adapter.Ensure that the plate barcode and serial number are facing the front of theinstrument.

2. Remove the clear plastic sheet from the outside of the plate lid.

3. Touch to retract the loading arm.

4. In the Home screen of the QuantStudio™ 12K Flex Software, in the Run pane,click OpenArray.

5. In the Select Instrument pane, select your QuantStudio™ instrument.

6. Click Get Plate IDs to import the barcodes of the OpenArray™ plates.

Chapter 3 Prepare and run TaqMan® Advanced miRNA Assays with fixed-content OpenArray™ platesRun the OpenArray™ plate(s) on the QuantStudio™ 12K Flex instrument 3


7. Click Import, then Browse to import the EDS file created in “GenerateOpenArray™ plate layouts in the QuantStudio™ 12K Flex Software“ on page 21.

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. Waitfor the system to complete the run before opening the EDS file.

11. Check the QC images for loading issues or leaks.

Check the QC images

Check the QC images before analysis. For additional information, see Appendix A,“Troubleshooting“.

1. In the QuantStudio™ 12K Flex Software Export screen:a. Click Browse to create a uniquely-named folder for the QC images export.

b. Click Export QC Images (bottom of screen).

IMPORTANT! Create a new folder for images each time; exporting a second runto the same folder overwrites the images.

2. View the following ROX™ image to check for loading quality issues:• POST-READ_CHANNEL_4.tiff

3. Check for leaks or other displaced sample issues.a. View the following spotfinding images:

• s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff• s02_c040_t03_p0001_m1_x2_e1_cp#_spotfind.tiff

Note: The “cp#” in the image file name refers to the array position (1–4)within the instrument.

b. If a problem is found, view the following pre-run spotfinding image todetermine if the issue existed even before cycling (this is useful fortroubleshooting):

• s00_c001_t01_p0001_m2_x3_e1_cp#_spotfind.tiff

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

• STAGE2_CYCLE1_CHANNEL_1.tiff• STAGE2_CYCLE40_CHANNEL_1.tiff

5. Note any abnormalities found, as well as all other potentially relevantinformation related to the setup of the run.

Chapter 3 Prepare and run TaqMan® Advanced miRNA Assays with fixed-content OpenArray™ platesCheck the QC images3


One-time procedures

1. Go to thermofisher.com/OA-platefiles.

2. From the Select Your Product dropdown list, select TaqMan® OpenArray™

Inventoried (PGx, miRNA, Pathway), Training & Endogenous Control Panels.

3. In the OpenArray™ templates (EDTs) for panels tab, click Download the EDTfiles.

4. Save the appropriate EDT file.

Download EDTfiles

Chapter 3 Prepare and run TaqMan® Advanced miRNA Assays with fixed-content OpenArray™ platesOne-time procedures 3



Prepare and run TaqMan® AdvancedmiRNA Assays with flexible-contentand custom-configured OpenArray™

plates

■ Generate 384-well sample plate layouts in the OpenArray™ SampleTracker Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

■ Set up the PCR reactions in an OpenArray™ 384-well Sample Plate . . . . . . . . . 32

■ Set up the AccuFill™ instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

■ Transfer reactions to the OpenArray™ plate (AccuFill™ instrument) . . . . . . . . . 34

■ Seal the OpenArray™ plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

■ Run the OpenArray™ plate(s) on the QuantStudio™ 12K Flex instrument . . . . 36

■ Check the QC images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

■ One-time procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

The workflow for flexible-content and custom-configured OpenArray™ plates usesOpenArray™ Sample Tracker Software to generate the plate layouts.

4


Generate 384-well sample plate layouts in the OpenArray™ SampleTracker Software

For flexible-content and custom-configured OpenArray™ plates, use the OpenArray™

Sample Tracker Software to map assays and samples onto the TaqMan® OpenArray™

Plates.

The software maps the 1:20 dilution of the miR-Amp reaction product to a 384-wellsample plate layout as CSV files that are used by OpenArray™ AccuFill™ software.

Before generating a plate layout, see page 38 and page 39 to:• Set up optimized folder locations and software preferences.• Download the TPF file(s) for the OpenArray™ plate(s).

1. Generate the 96-well sample plate CSV file using the 96-Well Sample Plate 1.csvtemplate, then save it to the Sample Tracker 96-well Input folder.The 96-Well Sample Plate 1.csv file is provided in the AccuFill™ softwareinstallation. Enter or copy the sample names in 96-Well Sample Plate 1.csv, thenSave as a new CSV-format file.

2. In the Sample Tracker Software Properties screen, select Gene Expression forExperiment Type, then select the appropriate settings for OpenArray™ Plate andPipettor.

3. In the Samples screen, click Import, then select and import the sample CSVfile.

4. In the Sample Mapping screen, confirm that the samples for a singleOpenArray™ plate are assigned to one color.If necessary, correct the OpenArray™ Plate and Pipettor settings in the Propertiesscreen.

5. In the Sample Mapping screen, click the 384-Well Plate tab, then clickExport4Export *.csv.

6. Select 384-Well Plate (for AccuFill), then save the exported file.

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

Chapter 4 Prepare and run TaqMan® Advanced miRNA Assays with flexible-content and custom-configuredOpenArray™ plates

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

4


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

IMPORTANT! The 4 × 12 area(s) of the 384-well plate being filled must match thearea(s) designated in the OpenArray™ Sample Tracker Software for that set ofsamples.

1. Remove the OpenArray™ plate from the freezer; allow it to come to roomtemperature in its unopened sleeve (~15 minutes).The OpenArray™ plate must be completely thawed before transferring reactionsto it from the 384-well sample plate.

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

3. Combine master mix and cDNA samples in tubes, strip tubes, or a 96-well plate.

Component

OpenArray™ Plate Format

18 and 56 112 168 224

Vol.[1] Vol.[2,3] Vol.[2,4] Vol.[2,5]

TaqMan®

OpenArray™

Real-Time PCRMaster Mix

2.5 µL 6.3 µL 9.4 µL 12.5 µL

Diluted cDNAtemplate

2.5 µL 6.3 µL 9.4 µL 12.5 µL

Total reactionvolume

5.0 µL 12.6 µL 18.8 µL 25.0 µL

[1] Volume per sample, 1 sample per subarray.[2] Volumes include 25% overage.[3] Volume per sample, 1 sample per 2 subarrays.[4] Volume per sample, 1 sample per 3 subarrays.[5] Volume per sample, 1 sample per 4 subarrays.

4. Following the plate layout designated in the OpenArray™ Sample TrackerSoftware, add 5.0 µL of the combined master mix and cDNA sample to theappropriate well in the OpenArray™ 384-well Sample Plate.

1

2

1 One full array2 One subarray

5. Thoroughly mix each PCR reaction by pipetting up and down or by using the"mix" function on a multi-channel pipette.

Chapter 4 Prepare and run TaqMan® Advanced miRNA Assays with flexible-content and custom-configuredOpenArray™ platesSet up the PCR reactions in an OpenArray™ 384-well Sample Plate

4


6. Seal the plate with an aluminum foil seal, remove the foil flap, then mark theedges of the filled 4 × 12 area with a pen.

7. Centrifuge the plate at 1,000 rpm for 1 minute.

8. Score the foil along the lines that were marked before centrifuging.Do not remove the foil from the scored area at this time.


IMPORTANT! Do not use OpenArray™ AccuFill™ System Tips that exceed theexpiration date (shown on the outer box that contains the tip trays).

1. In the OpenArray™ AccuFill™ software, click Setup and Load.

2. In the Setup Load Information window, ensure that the Use Sample Integrationcheckbox is selected.

2

1

1 Use Sample Integration checkbox2 Browse buttons

3. Click Browse to the right of Plate Holder Position, then select the 384-wellsample plate CSV file that was generated with the OpenArray™ Sample TrackerSoftware.The Browse buttons appear after the Use Sample Integration is selected.

4. Click Browse to the right of the Plate Holder Position corresponding to theOpenArray™ of interest, then select the TPF file corresponding to the desiredOpenArray™ plate.



4


5. Click the corresponding 4 × 12 area of the 384-well plate, then click Next to openthe Setup Deck window.

6. Ensure that:• Tip boxes are loaded in the AccuFill™ instrument as illustrated in the

following figure:

• Lids are removed from the tip boxes.• The waste bin in the instrument is emptied.

7. In the Setup Deck window, select:• The tips are configured as shown above• The Waste Bin is empty

Transfer reactions to the OpenArray™ plate (AccuFill™ instrument)

Ensure that the OpenArray™ plate is thawed and that the entire plate is at roomtemperature.

1. Prepare the items needed to seal the OpenArray™ plate (next section).

Note: The OpenArray™ plate must be sealed promptly after being loaded withthe reactions (this section).

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

b. Gather and remove from packaging an OpenArray™ lid, plug, syringe withOpenArray™ Immersion Fluid, and syringe tip.

c. Attach the syringe tip to the syringe and carefully push some of the fluidthrough the tip to remove air bubbles, then lay the syringe aside.

2. Remove the OpenArray™ plate from its sleeve and place it in the plate holder ofthe AccuFill™ instrument.Ensure that the bar code on the OpenArray™ plate is facing left and the serialnumber is facing right.

3. Using forceps, peel the foil from the filled area of the OpenArray™ 384-wellSample Plate.

4. Close the instrument door.

Chapter 4 Prepare and run TaqMan® Advanced miRNA Assays with flexible-content and custom-configuredOpenArray™ platesTransfer reactions to the OpenArray™ plate (AccuFill™ instrument)

4


5. In the AccuFill™ software Setup Deck window, select the followingconfirmations, then click Load.

• The OpenArray Plate is in the Plate Holder• Remove foil from the highlighted section of the Sample Plate

6. As soon as the Remove OpenArray Plate window appears, open the instrumentdoor, then remove the loaded OpenArray™ plate.

7. Proceed immediately to seal the OpenArray™ plate (next section).

Note: For best results, seal the OpenArray™ plate within 90 seconds ofcompletion of loading, to prevent evaporation.


IMPORTANT! Handle the OpenArray™ plate and case only by the edges throughoutthis procedure.

1. Place the filled OpenArray™ plate in the QuantStudio™ 12K Flex OpenArray™

Plate Press 2.0.Ensure that the bar code is facing left and the serial number is facing right.

2. Remove the clear plastic sheet fromthe inside of the lid, remove the redprotective film around the edge of theOpenArray™ lid, then seat the lid onthe OpenArray™ case in the platepress.

3. Engage the press mechanism until thegreen flashing light changes to asteady green light (~20 seconds).

4. Disengage the press, then remove theOpenArray™ case.

2

1

4

3

1 Protective film (remove)2 Adhesive3 Protective film (remove)4 Notched end (align with serial number)



4


5. While holding the OpenArray™ case by the edges, insert the prepared syringe tipinto the port in the case, then carefully inject immersion fluid until the case isfilled.

Note: Minimize creation of air bubbles when you dispense the fluid; one smallair bubble in the case is acceptable.

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

6. While holding the case vertically, remove the syringe tip, insert the screw end ofthe OpenArray™ plug into the port and rotate clockwise until the black handlebreaks off.

IMPORTANT! To avoid leaking of immersion fluid, hold the case vertically androtate the plug slowly.

If the plug handle falls of prematurely, use a Phillips #0 screwdriver to completethe step.

7. Clean the case with a laboratory wipe that has been thoroughly sprayed withethanol, then dry the case with a clean laboratory wipe.

Run the OpenArray™ plate(s) on the QuantStudio™ 12K Flexinstrument

1. On the instrument touchscreen, touch to extend the loading arm, then placethe OpenArray™ plates on the plate adapter.Ensure that the plate barcode and serial number are facing the front of theinstrument.

2. Remove the clear plastic sheet from the outside of the plate lid.

3. Touch to retract the loading arm.

4. In the Home screen of the QuantStudio™ 12K Flex Software, in the Run pane,click OpenArray.

5. In the Select Instrument pane, select your QuantStudio™ instrument.

Chapter 4 Prepare and run TaqMan® Advanced miRNA Assays with flexible-content and custom-configuredOpenArray™ platesRun the OpenArray™ plate(s) on the QuantStudio™ 12K Flex instrument

4


6. Click Get Plate IDs to import the barcode(s) of the OpenArray™ plate(s).Once the OpenArray™ serial numbers appear, the loaded TPF files correspondingto each plate should appear in the Setup File field.If not, click Browse, then select the correct loaded TPF file from the Loaded TPFfolder.

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

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

9. Click Start Run.

Note: The instrument pauses at 41 or 42 seconds prior to the end of the run. Waitfor the system to complete the run before opening the EDS file.

10. Check the QC images for loading issues or leaks.

Check the QC images

Check the QC images before analysis. For additional information, see Appendix A,“Troubleshooting“.

1. In the QuantStudio™ 12K Flex Software Export screen:a. Click Browse to create a uniquely-named folder for the QC images export.


IMPORTANT! Create a new folder for images each time; exporting a second runto the same folder overwrites the images.

2. View the following ROX™ image to check for loading quality issues:• POST-READ_CHANNEL_4.tiff

3. Check for leaks or other displaced sample issues.a. View the following spotfinding images:

• s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff• s02_c040_t03_p0001_m1_x2_e1_cp#_spotfind.tiff

Note: The “cp#” in the image file name refers to the array position (1–4)within the instrument.

b. If a problem is found, view the following pre-run spotfinding image todetermine if the issue existed even before cycling (this is useful fortroubleshooting):

• s00_c001_t01_p0001_m2_x3_e1_cp#_spotfind.tiff

Chapter 4 Prepare and run TaqMan® Advanced miRNA Assays with flexible-content and custom-configuredOpenArray™ platesCheck the QC images

4


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

• STAGE2_CYCLE1_CHANNEL_1.tiff• STAGE2_CYCLE40_CHANNEL_1.tiff

5. Note any abnormalities found, as well as all other potentially relevantinformation related to the setup of the run.

One-time procedures

This procedure simplifies the file locations used in the OpenArray™ AccuFill™

instrument software.

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 computerdrive as the AccuFill™ software:

• TPF Files• Sample Tracker 96-well Input• Sample Tracker 384-well CSV Files• Loaded TPF Files

2. (Optional) Navigate to <drive>:\Program files(x86)\AppliedBiosystems\OpenArray Sample Tracker\Examples, copy the96-Well Sample Plate 1.csv file, then paste it in the Sample Tracker 96-well Inputfolder.

3. In the OpenArray™ Sample Tracker Software, select View4Preferences, thenenter the following preferences:

Field Selection

Experiment Type Gene Expression

OpenArray™ Plate Select the OpenArray™ format that will be runmost often; for example, Gene Expression – 56

Pipettor Fixed or Adjustable

Import Data Directory Sample Tracker 96-well Input

Export Data Directory Sample Tracker 384-well CSV Files

4. In the AccuFill™ software, select Instrument4Edit Preferences, then:a. Select Require Sample Integration.

Set up defaultfolders andsoftwarepreferences

Chapter 4 Prepare and run TaqMan® Advanced miRNA Assays with flexible-content and custom-configuredOpenArray™ platesOne-time procedures

4


b. Select the indicated folders.

AccuFill™ folder Default folder Folder contents

OpenArray™ PlateFile Input Folder

TPF Files TPF files for theOpenArray™ plates; containassay name and location

Sample Plate FileFolder

Sample Tracker 384-wellCSV Files

CSV 384-well sample platelayout files

Loaded OpenArray™

Plate File FolderLoaded TPF Files Integrated TPF files that

are generated duringprocessing with theAccuFill™ software.

5. In the QuantStudio™ 12K Flex instrument software, selectTools4Preferences4OpenArray, then select the Loaded TPF Files folder for thesoftware Setup Folder.

Note: If the instrument software is not on the same computer as the AccuFill™

software, transfer the loaded TPF files to the computer running the instrumentsoftware.

Set up the optimized folder locations and software preferences before downloadingTPF files. See page 38.

To download TPF files for custom OpenArray™ plates, you need the Lot# and theSerial# from the packaging of each OpenArray™ plate.

1. Go to thermofisher.com/OA-platefiles.

2. From the Select Your Product dropdown list, select TaqMan® OpenArray™

Custom Gene Expression/Genotyping Plates.

3. Select a download option:

• I want to download all available TPF & AIF files• I want to download a specific TPF file

4. Enter the Lot# and the Serial#, then click Submit.

Note: The Serial# is case-sensitive.

5. Save the TPF files to the desktop TPF Files folder.

Note: Do not create sub-folders in the TPF Files folder. The software cannotaccess sub-folders.

Download TPFfiles

Chapter 4 Prepare and run TaqMan® Advanced miRNA Assays with flexible-content and custom-configuredOpenArray™ platesOne-time procedures

4



Export and review TaqMan®

Advanced miRNA Assay data

■ Export data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

■ Prepare exported data for analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

■ Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Export data

1. Open an EDS file in the QuantStudio™ 12K Flex Software.

2. In the Experiment Menu pane, click Export.

3. Click Load Export Set (bottom of the screen), select GE_export_setting, then clickOK.

4. Select .xlsx from the File Type dropdown list (top-right of the screen).

5. (Optional) Perform any of the following actions to customize the file export:

• Select Open file(s) when export is complete.• Click Browse to select a new Export File Location.• Enter a new file name in the Export File Name text field.• Click the Results tab, then select the content to export.

6. Click Start Export (bottom of the screen).If Open file(s) when export is complete is selected, then the file automaticallyopens. If the option is not selected, navigate to and open the exported XLSX file.

Prepare exported data for analysis

1. Open the exported XLSX data file.

2. Ensure that the barcode, run conditions, and all selected data columns wereexported correctly.

3. Scroll down to the data rows, select the headers and data, then copy-paste into anew worksheet.

4. Rename the new worksheet Data Table_Run File Name.

5


5. (Optional) To combine data from multiple OpenArray™ plates:a. Insert a Barcode column in the Data Table worksheet to track OpenArray™

barcodes.

b. Copy-paste the barcode numbers to the appropriate cells in the newBarcode column.

6. Find-replace all "Undetermined" values with an empty cell (no value) in the Crtcolumn.This step ensures an exact count of Crt values.

7. Delete rows that do not contain run data.

Review results

Note: These guidelines apply to results from experiments that included three or moretechnical replicates.

Note: We encourage testing and establishing your own Crt cut-off value for eachassay to achieve high sensitivity and specificity.

1. Review the exported data for through-hole results that may require specialattention.

2. Consider filtering out from analysis through-holes with the following values:

Parameter toexamine Consider filtering out through-holes if…

Crt Crt >28

Amp Score Amp Score <1.24

Note: Through-holes with unexpected Crt values can also be identified byreviewing the Amplification Plot.

3. Review through-holes with Crt >28 and ensure that the Crt values arereproducible in all technical replicates.

Note: Crt=28 is approximately equal to 1 copy of the target sequence in areaction.

4. Take note of technical replicates with mean Crt <28 and a high standarddeviation (>0.5). The data from these through-holes may require further review.

5. Ensure that at least two of the three replicates amplified adequately and passyour review specifications.

6. Use your preferred method to analyze the data.

Chapter 5 Export and review TaqMan® Advanced miRNA Assay dataReview results 5


To review results using pivot tables, you can use the following settings.

Note: For the "Average of" and "StdDev of" summarizations, use the appropriatesource field (Crt or Amp Score), then choose the calculation type.

Area of pivot tableFields to add

Target-oriented view Sample-oriented view

Report Filter — Sample Name[1]

Column Labels Sample Name —

Row Labels Target Name Target Name

Values Average of Crt Average of Crt

StdDev of Crt[2] StdDev of Crt

Count of Crt Count of Crt

— Average of Amp Score

[1] To see individual sample results, select the sample from the dropdown list next to the Sample Name header.[2] A Values field will automatically appear in the Column Labels area.

Fields for PivotTables

Chapter 5 Export and review TaqMan® Advanced miRNA Assay dataReview results5


Troubleshooting

Troubleshoot with cycling and imaging run images (QC images)

Many problems with OpenArray™ results can be diagnosed by examining the qualitycontrol (QC) images taken at various points during a cycling/imaging run.

The QC images are fluorescent or reflected light images taken before, during, andafter cycling. They may require adjustment to make image features visible. To viewthe images, we recommend that you install the free software program ImageJ, whichallows you to easily manipulate the images in ways that other image viewers cannot.

1. In the QuantStudio™ 12K Flex Software Export screen:a. Click Browse to select a uniquely-named folder for the QC images export.


IMPORTANT! Select a new folder for images each time; exporting a second runto the same folder overwrites the images.

2. Use ImageJ to view the images of interest.

To... View image... Image description

Confirm the identity ofimages within a folder

BARCODE IMAGE.tiff Reflected light image of the entireOpenArray™ plate

Evaluate the loadingquality

PRE-READ_CHANNEL_4.tiffPOST-READ_CHANNEL_4.tiff

Pre- and post-ROX™ images

Check for existingcontamination on thecase and/or heatedcover

s00_c001_t01_p0001_m2_x3_e1_cp#_spotfind.tiff[1] Pre-run reflected light spotfindingimage (used by the software todetermine the location of the holes)

Identify potential leaksor other contamination

s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff[1] Mid-run reflected light spotfindingimage

s02_c040_t03_p0001_m1_x2_e1_cp#_spotfind.tiff[1] Post-run reflected light spotfindingimage

Look at patterns in thefluorescent data (forexample, gradients)

STAGEx_CYCLEy_CHANNEL_1.tiff FAM™ images at a particular cycle(y) of a particular stage (x) of therun.

[1] The “cp#” in the image file name refers to the array position (1–4) within the QuantStudio™ 12K Flex instrument.

3. (Optional) Adjust the images for brightness and/or contrast to make imagefeatures visible.

a. Open the image in ImageJ.

A


b. Select Image4Adjust Brightness/Contrast (or press Ctrl+Shift+C).

c. Click Auto or adjust the sliders until the features of interest in the image arevisible.

AccuFill™ instrument plate loading errors

Observation Possible cause Recommended action

There are empty through-holes Insufficient sample wasadded to the 384-well sampleplate.

Use proper pipettingtechniques. Ensure that thereare no air bubbles in thepipette tips after sampleaspiration.

Reaction mix (sample +master mix) is not at thebottom of the 384-wellsample plate.

Centrifuge the sample plateat 1,000 rpm for 60 seconds.

Turn-holes are repeatedly missed AccuFill™ instrument isaligned too far to the left or tothe right.

Systematic loading problemscan occur with the AccuFill™

instrument, which indicates aneed for service. For example,when turn-holes arerepeatedly missed acrossmultiple subarrays, service isrequired. Turn‑holes arewhere the AccuFill™

instrument changes directionduring sample loading.

Start pointsStop points

Turn holes

Contact your local fieldservice engineer.

Appendix A TroubleshootingAccuFill™ instrument plate loading errorsA



Entire subarrays are missing The sample/master mix wasnot added to particular wellsin the 384-well sample plate.

Visually inspect the sampleplate to ensure that the wellshave sample/master mix.

Stuck tip mandrel onAccuFill™ instrument mayneed cleaning.

Contact your local fieldservice engineer.

Pipette tip was not loaded onmandrel.

Contact your local fieldservice engineer for frequentoccurrences (infrequentoccurrences can be due to apoorly molded tip).

OpenArray™ plate assembly and handling errors


Case leaks and bubbles inside the case

Improper sealing of theOpenArray™ plate in theOpenArray™ Case can lead to immersion fluidleaks or bubble formation inside the case, leadingto uneven heating and imaging throughout PCRand to poor quality data.

The images above are examples of OpenArray™

plates that have been affected by immersion fluidleaks. The images show where leaked fluid hascondensed on the underside of the heated coverwindows and obscured the view of the through‐holes.

Plate press was not engagedfor at least 20 seconds.

Fully engage the plate pressfor at least 20 seconds.

Damaged lid adhesive. Remove the liner and visuallyinspect the lid adhesives fordefects. Ensure that adhesiveis not damaged or warped.

Damaged fill port screwgasket.

Visually inspect the screw toensure that the orange gasketis present and not damaged.

Damaged fill port screwassembly. Breaks off tooeasily.

The screw may be mis-threaded: unscrew it and usea new screw assembly.

Oily lid or case fromimmersion fluid overflow.

Wipe off excess overflow ofimmersion fluid from the lid,case bottom, and creviceswith 70% isopropyl alcohol,using a lint-free cloth (thecloth included with theOpenArray™ plate isacceptable).

Immersion fluid was exposedto air for too long.

Do not remove the immersionfluid syringe cap or draw airbubbles into the syringe untilyou are ready to load.

Appendix A TroubleshootingOpenArray™ plate assembly and handling errors A


Observation Possible cause Recommended actionThe best image in which to detect leaks is thes02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiffimage. This image is taken at the start of cycling,which is where most leaks occur. See “Troubleshoot with cycling and imaging runimages (QC images)“ on page 43.

Too large of a bubble insidethe OpenArray™ case aftersealing.

Minimize the size of thebubble by tilting theOpenArray™ case so that thefill port is at the highest point.Slowly fill the case withimmersion fluid until only asmall air bubble remains.Attach the screw and wipe offany excess oil that may havespilled onto the case.

Damaged plate press, leadingto uneven pressure.

Contact your field serviceengineer if you suspect thatyour plate press may bedamaged.

Sample blow-out during the addition of immersionfluid

The reactions in A12 werecompromised during theaddition of immersion fluid.Injecting the immersion fluidtoo quickly can purge thesample out of the through-holes near the fill port. Oftenthis is caused by the user notpurging the syringe slightlybefore use.

Dispense a small amount ofimmersion fluid onto a papertowel before use to ensuresmooth operation of thesyringe.

Evaporation of reaction mixture in through-holes Too much time elapsed beforethe plate was sealed with lidand immersion fluid. In thisexample, the top half of eachsubarray was intentionally leftopen to the environment todemonstrate the effect ofevaporation. “Donuts” are aresult of the evaporated fluidin the though-holes.

Add immersion fluid as soonas the case is removed fromthe plate press to minimizethe likelihood of evaporation,then seal the case with thelid.

Appendix A TroubleshootingOpenArray™ plate assembly and handling errorsA


Supplemental information

■ Endogenous and exogenous controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

■ Overview of cDNA template preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

■ Overview of TaqMan® Advanced miRNA Assays chemistry . . . . . . . . . . . . . . . 49

■ Best practices for PCR and RT-PCR experiments . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Endogenous and exogenous controls

An endogenous control shows gene expression that is relatively constant andmoderately abundant across treatment protocols, and tissues or cell types.Normalization to endogenous control genes is currently the most accurate method tocorrect for potential biases that are caused by:

• Sample collection• Variation in the amount of starting material• Reverse transcription (RT) efficiency• Nucleic acid (RNA/DNA) preparation and quality

No single control can act as a universal endogenous control for all experimentalconditions, so we recommend verifying the chosen endogenous control or set ofcontrols for the sample tissue, cell, or treatment.

See “Endogenous and exogenous controls“ on page 14 for available TaqMan®

Advanced miRNA Assays that target miRNAs with relatively constant expressionlevels across many different sample types.

An exogenous control is a synthetic RNA oligonucleotide with an miRNA targetsequence that is not present in the sample of interest. For example, the target sequencefor the miRNA assay ath-miR-159a is not present in humans, so it is a good exogenouscontrol for human samples.

The RNA oligonucleotide is combined with the biological sample during the RNAisolation procedure as a spike-in control to monitor:

• Sample input amount for difficult samples (for example, serum/plasma or otherbiofluids).

• Extraction efficiency.

When using exogenous controls with TaqMan® Advanced miRNA Assays:• The assay chemistry requires that exogenous controls be 5'-phosphorylated.• The final concentration of the spike-in control in the sample should be 1–10 pM.

See Table 7 on page 14 for available TaqMan® Advanced miRNA Assays which targetsequences that can be used as exogenous controls with human samples.

B

Endogenouscontrols

Exogenouscontrols


Overview of cDNA template preparation

Quantification using TaqMan® Advanced miRNA Assays requires the modification ofmature miRNAs by the addition of a poly(A) tail (3’) and an adaptor (5’) to:

• Amplify all miRNAs in a single reverse transcription (RT) reaction.• Amplify the sample for downstream PCR in a single universal cDNA reaction.

5' AAAAAAAAAAAA 3'

Poly(A) tailmiRNA

PAPPoly(A) tailing reaction

Starting with a total RNA sample, poly(A)polymerase is used to add a 3’‑adenosine tailto the miRNA.

AAAAAAAAAAAA 3'Lig

5'

Adaptor

Adaptor ligation reaction

The miRNA with poly(A) tail undergoesadaptor ligation at the 5’ end. The adaptor actsas the forward-primer binding site for themiR-Amp reaction.

AAAAAAAAAAAART (V)TTTTTTTTTTTT

Universal RT primer

5' 3' Reverse transcription (RT) reaction

A Universal RT primer binds to the 3’ poly(A)tail and the miRNA is reverse transcribed. Theresulting cDNA is suitable for all TaqMan®

Advanced miRNA Assays.

miR-AMPforward primer

TTTTTTTTTTTT 5'3'P

miR-AMPreverse primer

P5' 3'

miR-Amp reaction

Universal forward and reverse primersincrease the number of cDNA molecules.

LEGEND:

Hot-startDNA polymerasePReverse

TranscriptaseRTLigaseLigPoly(A)polymerasePAP

Appendix B Supplemental informationOverview of cDNA template preparationB


Overview of TaqMan® Advanced miRNA Assays chemistry

TaqMan® MGB probes contain:• A reporter dye (for example, FAM™) at the 5′ end of the probe.• A non-fluorescent quencher (NFQ) dye at the 3′ end of the probe.

The NFQ dye does not fluoresce, which allows the real-time PCR system tomeasure the reporter dye contributions more accurately.

• A minor groove binder (MGB) at the 3´ end of the probe that:– Increases the melting temperature (Tm) without increasing the probe length.– Allows for the design of shorter probes.

Note: The following figures are general representations of real-time PCR withTaqMan® MGB probes and TaqMan® Advanced miRNA Assays. The sequence regionsare not necessarily drawn to scale.

The 5' nuclease assay process takes place during PCR amplification. It occurs in everycycle and does not interfere with the exponential accumulation of product.

Poly(A) tail regionAdaptor region miRNA region

3' 5'5' 3'

Figure 1 cDNA synthesis product

During the PCR, the forward and reverse primers anneal to complementary sequencesalong the denatured cDNA template strands (Figure 2). The primer binding sites varydepending on the target miRNA sequence and are designed to maximize specificity. Figure 2 shows an example representation in which the reverse primer is the primerthat partially overlaps the miRNA region.

The TaqMan® MGB probe anneals specifically to a complementary sequence betweenthe forward and reverse primer sites (Figure 2). When the probe is intact, theproximity of the reporter dye and quencher dye suppresses the reporter fluorescence,primarily by Förster-type energy transfer.

R NFQ

MGBProbe

Reverse primer

Forward primer

3' 5'

5' 3'

Figure 2 Annealing of probes and primers to cDNA strands

TaqMan® MGBprobes

About the 5'nuclease assay

Appendix B Supplemental informationOverview of TaqMan® Advanced miRNA Assays chemistry B


During polymerization, the DNA polymerase cleaves only probes that hybridize tothe target sequence. Cleavage separates the reporter dye from the probe. Theseparation of the reporter dye from the quencher dye results in increased fluorescenceby the reporter dye (Figure 3).

This increase in fluorescence occurs only if the probe is complementary to the targetsequence and if the target sequence is amplified during PCR. Because of theseconditions, nonspecific amplification is not detected.

NFQ

MGBR

P3' 5'

5' 3'P

Figure 3 Initial polymerization and cleavage of reporter dye

Polymerization of the strand continues (Figure 4), but because the 3' end of the probeis blocked, no extension of the probe occurs during PCR.

R

NFQ

MGB

3' 5'

5' 3'

Figure 4 Completion of polymerization

LEGEND:

Hot-startDNA polymeraseP Reporter dye Non-fluorescent

quencher dyeNFQ Minor groovebinderMGBR

Appendix B Supplemental informationOverview of TaqMan® Advanced miRNA Assays chemistryB


Best practices for PCR and RT-PCR experiments

When preparing samples for PCR or RT-PCR amplification:• Wear clean gloves and a clean lab coat.

– Do not wear the same gloves and lab coat that you have previously usedwhen handling amplified products or preparing samples.

• Change gloves if you suspect that they are contaminated.• Maintain separate areas and dedicated equipment and supplies for:

– Sample preparation and reaction setup.– Amplification and analysis of products.

• Do not bring amplified products into the reaction setup area.• Open and close all sample tubes carefully. Avoid splashing or spraying samples.• Keep reactions and components capped as much as possible.• Use a positive-displacement pipettor or aerosol-resistant barrier pipette tips.• Clean lab benches and equipment periodically with 10% bleach solution or DNA

decontamination solution.

Fluorescent contaminants can generate false positive results. To help detect thesecontaminants, we recommend including a no-amplification control reaction thatcontains sample, but no Master Mix.

After PCR, if the absolute fluorescence of the no-amplification control is greater thanthe fluorescence of the no template control (NTC), fluorescent contaminants may bepresent in the sample or in the heat block of the real-time PCR instrument.

Good laboratorypractices for PCRand RT-PCR

Detect fluorescentcontaminants

Appendix B Supplemental informationBest practices for PCR and RT-PCR experiments B


Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specifiedin the user documentation may result in personal injury or damage to theinstrument or device. Ensure that anyone using this product has receivedinstructions in general safety practices for laboratories and the safetyinformation provided in this document.

· Before using an instrument or device, read and understand the safetyinformation provided in the user documentation provided by themanufacturer of the instrument or device.

· Before handling chemicals, read and understand all applicable Safety DataSheets (SDSs) and use appropriate personal protective equipment (gloves,gowns, eye protection, etc). To obtain SDSs, see the “Documentation andSupport” section in this document.

C


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,ensure laboratory personnel read and practice the general safety guidelines forchemical usage, storage, and waste provided below. Consult the relevant SDSfor specific precautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by thechemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, see the “Documentation andSupport” section in this document.

· Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer's cleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.· Ensure use of primary and secondary waste containers. (A primary waste

container holds the immediate waste. A secondary container contains spillsor leaks from the primary container. Both containers must be compatiblewith the waste material and meet federal, state, and local requirements forcontainer storage.)

· After emptying a waste container, seal it with the cap provided.· Characterize (by analysis if necessary) the waste generated by the particular

applications, reagents, and substrates used in your laboratory.· Ensure that the waste is stored, transferred, transported, and disposed of

according to all local, state/provincial, and/or national regulations.· IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

Appendix C SafetyChemical safety C


Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. Conduct all work in properly equipped facilitieswith the appropriate safety equipment (for example, physical containmentdevices). Safety equipment can also include items for personal protection, suchas gloves, coats, gowns, shoe covers, boots, respirators, face shields, safetyglasses, or goggles. Individuals should be trained according to applicableregulatory and company/ institution requirements before working withpotentially biohazardous materials. Follow all applicable local, state/provincial,and/or national regulations. The following references provide generalguidelines when handling biological samples in laboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiologicaland Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)21-1112, Revised December 2009; found at:www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Appendix C SafetyBiological hazard safetyC


http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Documentation and support

Related documentation

Document Pub. No.

TaqMan® Advanced miRNA Assays Quick Reference—Fixed-content TaqMan® OpenArray™

PlatesMAN0016125

TaqMan® Advanced miRNA Assays Quick Reference—Flexible-content and custom-configured TaqMan® OpenArray™ Plates

MAN0017500

TaqMan® Advanced miRNA cDNA Synthesis Kit Product Information Sheet MAN0011141

A technical guide to identifying miRNA normalizers using TaqMan® Advanced miRNA Assays COL31302 0916[1]

QuantStudio™ 12K Flex Real-Time PCR System Maintenance and Administration Guide 4470689

QuantStudio™ 12K Flex Real-Time PCR System: Multi‐Well Plates and Array CardExperiments User Guide

4470050

OpenArray™ Sample Tracker Software Quick Reference 4460657

OpenArray™ AccuFill™ System User Guide 4456986

[1] Available at thermofisher.com/advancedmirna (in the Documentation section).



Customer and technical support

Visit thermofisher.com/support for the latest in services and support, including:• Worldwide contact telephone numbers• Product support, including:

– Product FAQs– Software, patches, and updates– Training for many applications and instruments

• Order and web support• Product documentation, including:

– User guides, manuals, and protocols– 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 affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale found on LifeTechnologies' website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact LifeTechnologies at www.thermofisher.com/support.

Documentation and supportCustomer and technical support


http://thermofisher.com/support

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/support

thermofisher.com/support | thermofisher.com/askaquestion

thermofisher.com

8 January 2018

http://thermofisher.com/support

mailto:thermofisher.com/askaquestion

http://thermofisher.com

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