megaplex primer pools - thermo fisher scientific...megaplex primer pools for microrna expression...

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

Megaplex™ Primer PoolsUSER GUIDE

For MicroRNA expression analysis

for use with TaqMan™ Array CardsPublication Number 4399721

Revision E

Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, CA 94566For 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: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. 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 YOUR USE OF IT.

Revision history: Pub. No. 4399721

Revision Date DescriptionE 10 December 2019 Removed troubleshooting and replaced with link to FAQs.

D 29 November 2018 • Updated available products.

• Added compatible real–time PCR instruments, thermal cyclers, and other applicableproducts.

• Updated information about how to obtain files for the cards.

• Added detailed instructions to prepare TaqMan™ Array Cards.

• Updated Master Mix recommended for real–time PCR.

• Updated instructions to set up and run the real–time PCR instruments.

• Updated guidelines to analyze data.

• Updated for general style, formatting, and branding.

C July 2010 Baseline for this revision history.

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. Eppendorf and MixMate are trademarks of Eppendorf AG.

©2019 Thermo Fisher Scientific Inc. All rights reserved.

https://www.thermofisher.com/symbols-definition

Contents

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

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

TaqMan™ Array Card overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

■ CHAPTER 2 Prepare the sample and select the workflow . . . . . . . . . . 14

Guidelines for total RNA input . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Select the workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■ CHAPTER 3 Perform Megaplex™ Primer Poolswith preamplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Guidelines for Megaplex™ reverse transcription with preamplification . . . . . . . . . . . . 15Guidelines for performing real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Perform Megaplex™ reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Perform the preamplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Dilute the preamplification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Perform PCR amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Prepare PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Prepare a TaqMan™ Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Set up and run the real–time PCR instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Megaplex™ Primer Pools for MicroRNA expression analysis User Guide 3

■ CHAPTER 4 Perform Megaplex™ Primer Poolswithout preamplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Guidelines for Megaplex™ reverse transcription without preamplification . . . . . . . . . 21Guidelines for performing real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Perform Megaplex™ reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Perform PCR amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Prepare PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Prepare a TaqMan™ Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Set up and run the real–time PCR instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

■ CHAPTER 5 Analyze data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Algorithms for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Data normalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Megaplex™ assay performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Megaplex™ Assay Performance File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

■ APPENDIX A Troubleshooting and FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

■ APPENDIX B Detailed procedures for preparation of aTaqMan™ Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Guidelines for preparation of a card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

TaqMan™ Array Card diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Load the PCR reaction mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Centrifuge the card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Seal the card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

■ APPENDIX C Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

How to import setup files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Setup files for compatible instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Megaplex™ Primer Pools chemistry overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Preamplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39TaqMan™ MGB probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40About the 5' nuclease assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Best practices for PCR and RT-PCR experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Detect fluorescent contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Contents

4 Megaplex™ Primer Pools for MicroRNA expression analysis User Guide

■ APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Contents


Product information

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

■ TaqMan™ Array Card overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

■ Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■ Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Product description

This document is about TaqMan™ MicroRNA Assays.

Note: See thermofisher.com/advancedmirna for more information about TaqMan™

Advanced miRNA Assays.

Applied Biosystems™ Megaplex™ RT Primers streamline the workflow forquantitating up to 380 MicroRNAs in parallel by enabling reverse transcription ofRNA to cDNA for all MicroRNAs in a single reaction. A companion pool ofMegaplex™ PreAmp Primers enables an optional preamplification step to be added tothe workflow when the sample is limiting or sensitivity is important. Megaplex™ RTPrimers and Megaplex™ PreAmp Primers are available for both human and rodent(mouse and rat) species.

Real-time PCR can be performed on the cDNA with fixed-content TaqMan™

MicroRNA Assays on TaqMan™ Array Cards or with individual TaqMan™ MicroRNAAssays.

• Megaplex™ RT Primers—A set of two predefined pools (Pool A and Pool B) of upto 380 stem-looped reverse-transcription (RT) primers per pool that enable thesimultaneous synthesis of cDNA for mature miRNAs.

• Megaplex™ PreAmp Primers (Optional)—A set of two pools (Pool A and Pool B)of gene-specific forward and reverse primers intended for use with very smallquantities of starting material. The primers enable the unbiased preamplificationof the miRNA cDNA target by PCR prior to loading the TaqMan™ MicroRNAAssay.

• TaqMan™ MicroRNA Assay—Dried-down TaqMan™ primers and probes. TheTaqMan™ Array Card enables quantitation of gene expression levels of up to 380miRNAs and controls. cDNA, with or without preamplification, is loaded ontothe card for real-time PCR.

See thermofisher.com/openarray for more information about TaqMan™ OpenArray™

Plates with TaqMan™ MicroRNA Assays.

1


http://www.thermofisher.com/advancedmirna

http://www.thermofisher.com/openarray

TaqMan™ Array Card overview

TaqMan™ Array Cards are 384-well microfluidic cards that are prepared with dried-down TaqMan™ Assays. With array cards, gene expression is measured using thecomparative Ct (ΔΔCt) method of relative quantitation. For more information aboutrelative quantitation, see the Resources section at thermofisher.com/taqman.

Advantages of using TaqMan™ Array Cards include:• Small-volume design that minimizes sample and reagent consumption.• Streamlined reaction setup that saves time and reduces labor-intensive steps.• Access to high-throughput, 384-well format without liquid-handling robotics.• Two-fold discrimination detection at the 99.7% confidence level.• Standardization across multiple samples in multiple laboratories.

Each card can run 1 to 8 samples against 12 to 384 TaqMan™ Assay targets (includingcontrols).

87

65

43

21

3

12

4

1 Fill reservoir—Each reservoir is loaded with a sample-specific PCR reaction mix; theassociated reaction wells fill with that sample (8 total reservoirs)

2 Fill reservoir strip—Support strip for fill reservoirs; removed before running the card3 Reaction well—Each well contains dried-down assay (384 total reaction wells)4 Reaction well row—A set of reaction wells that fill with the same sample-specific PCR

reaction mix (8 total rows, each row associated with a single fill reservoir)

Chapter 1 Product informationTaqMan™ Array Card overview 1


http://www.thermofisher.com/taqman

Contents and storage

Table 1 Megaplex™ Primer Pools sets

Contents Cat. No. Amount Storage

Megaplex™ Primer Pools, Human Pools Set v3.0

Includes:

• Megaplex™ RT Primers, Human Pool A v2.1

• Megaplex™ RT Primers, Human Pool B v3.0

• Megaplex™ PreAmp Primers, Human Pool A v2.1

• Megaplex™ PreAmp Primers, Human Pool B v3.0

4444750 50 reactions Store at −20°C

Megaplex™ Primer Pools, Rodent Pools Set v3.0

Includes:

• Megaplex™ RT Primers, Rodent Pool A

• Megaplex™ RT Primers, Rodent Pool B v3.0

• Megaplex™ PreAmp Primers, Rodent Pool A

• Megaplex™ PreAmp Primers, Rodent Pool B v3.0

4444766 50 reactions Store at −20°

Table 2 TaqMan™ MicroRNA Assays (TaqMan™ Array Cards)

Contents Cat. No. Amount Storage[1]

TaqMan™ Array Human MicroRNA A Cards v2.0 4398965 4 cards

Shipped at ambienttemperature.

Store at 2−8°C.

Protect from light.

TaqMan™ Array Human MicroRNA B Cards v3.0 4444910 4 cards

TaqMan™ Array Human MicroRNA A+B Cards Set v3.0 4444913 8 cards

TaqMan™ Rodent MicroRNA A Array v2.0 4398967 4 cards

TaqMan™ Array Rodent MicroRNA B Cards v3.0 4444899 4 cards

TaqMan™ Array Rodent MicroRNA A+B Cards Set v3.0 4444909 8 cards

[1] See packaging for expiration date.

Megaplex RT Primers and Megaplex PreAmp Primers can be purchased separately.

Table 3 Megaplex™ RT Primers


Megaplex™ RT Primers, Human Pool A v2.1 4399966

RT primers

MgCl2

50 reactions


Storeat −25°C to −15°C.

Megaplex™ RT Primers, Human Pool B v3.0 4444281

Megaplex™ RT Primers, Human Pool Set v3.0(Contains Pool A v2.1 and Pool B v3.0) 4444745

Megaplex™ RT Primers, Rodent Pool A 4399970

Chapter 1 Product informationContents and storage1



Megaplex™ RT Primers, Rodent Pool B v3.0 4444292 RT primers

MgCl2

50 reactions



Megaplex™ RT Primers, Rodent Pool Set v3.0(Contains Pool A and Pool B v3.0) 4444746


Table 4 (Optional) Megaplex™ PreAmp Primers


Megaplex™ PreAmp Primers, Human Pool A v2.1 4399233

50 reactions



Megaplex™ PreAmp Primers, Human Pool B v3.0 4444303

Megaplex™ PreAmp Primers, Human Pool Set v3.0(Contains Pool A V2.1 and Pool B v3.0 4444748

Megaplex™ PreAmp Primers, Rodent Pool A 4399203

Megaplex™ PreAmp Primers, Rodent Pool B v3.0 4444308

Megaplex™ PreAmp Primers, Rodent Pool Set v3.0(Contains Pool A and Pool B v3.0) 4444747


Go to thermofisher.com/taqmanfiles to download the following files:• Plate layout files (HTML and CSV formats)—Show the position of each assay on

the card. The HTML and CSV files contain identical information.• Setup files (SDS in TXT format)—Contain the sample setup on the card. Thefiles are imported into the software specific to your instrument to perform real-time PCR.

• Assay Information File (AIF in TXT format)—Describes the TaqMan™

MicroRNA Assays. See Understanding Your Shipment (Pub. No. MAN0017153) fordetailed information about the AIF.

• Assays Performance File (XLS format)—Indicates which assays show low no-template-control values. See “Megaplex™ Assay Performance File“ on page 28for more information.

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

Note: The run properties and the thermal protocol are not defined in the setup files.They must be set up in the instrument or software.

Chapter 1 Product informationContents and storage 1


https://www.thermofisher.com/taqmanfiles

Required materials not supplied

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

Table 5 Recommended products for isolation of RNA

Item Source

mirVana™ miRNA Isolation Kit, with phenol AM1560

mirVana™ miRNA Isolation Kit, without phenol AM1561

mirVana™ PARIS™ RNA and Native Protein PurificationKit AM1556

Table 6 Recommended products for preparation of cDNA

Item Source

TaqMan™ MicroRNA Reverse Transcription Kit 4366596

Table 7 Recommended preamplification Master Mix

Item Source

(Optional)TaqMan™ PreAmp Master Mix (2X) 4391128

Table 8 PCR Master Mix

Item Source

TaqMan™ Universal Master Mix II, no UNG 4440040

TaqMan™ Fast Advanced Master Mix 4444963

Table 9 Other materials and equipment required for the workflow

Item Source

Real-time PCR instrument, one of the following:

The instrument must be configured with the TaqMan™ Array Card block and heated cover.

QuantStudio™ 7 Flex Real-Time PCR System

Contact your localsales office

QuantStudio™ 12K Flex Real–Time PCR System

ViiA™ 7 Real-Time PCR System

7900HT Fast Real‑Time PCR System

Chapter 1 Product informationRequired materials not supplied1


http://www.thermofisher.com

http://fisherscientific.com

Item Source

Software

(Optional) Relative Quantification applicationAvailable at

thermofisher.com/cloud

(Optional) ExpressionSuite™ SoftwareAvailable at

thermofisher.com/expressionsuite

Equipment

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

• Veriti™ Thermal Cycler

• SimpliAmp™ Thermal Cycler

• ProFlex™ PCR System


Centrifuge with custom buckets and card holders,one of the following:

• Sorvall™ centrifuge

• Heraeus™ centrifuge

See the Resources section at thermofisher.com/taqmanarrays fora list of compatible centrifuges, rotors, and buckets.


TaqMan™ Array Card Sealer

(Referred to as Stylus Staker in some documents)Contact your local

sales office

Blank balance TaqMan™ Array Cards

(Included with the instrument block upgrade / installation kit)Contact your local

sales office

Microcentrifuge MLS

Vortex mixer MLS

(Optional) Eppendorf™ MixMate™ (shaker) Fisher-Scientific21-379-00

Pipettes MLS

Micropipettes MLS

Tubes, plates, and other consumables

Plastics consumables thermofisher.com/plastics

Aerosol-resistant barrier pipette tips MLS

Disposable gloves MLS

Chapter 1 Product informationRequired materials not supplied 1


https://www.thermofisher.com/us/en/home/cloud.html


http://www.thermofisher.com/expressionsuite


http://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-gene-expression/real-time-pcr-taqman-arrays.html?CID=fl-WE112784

http://www.thermofisher.com/plastics

http://www.thermofisher.com/plastics

Item Source

Reagents

Nuclease-free Water AM9938

TE, pH 8.0 AM9849

Chapter 1 Product informationRequired materials not supplied1


Workflow

Start with total RNA

▼

Select the workflow (page 14)

▼

Perform Megaplex™ Primer Poolswith preamplification

(page 15)(<8 hours)

Perform Megaplex™ Primer Poolswithout preamplification

(page 21)(<6 hours)

▼ ▼

Perform Megaplex™ reversetranscription

(page 16)(2.5 hours)

Perform Megaplex™ reversetranscription

(page 22)(2.5 hours)

▼ ▼

Perform the preamplification(page 17)(2 hours)

—

▼ ▼

Set up and run the real–time PCRinstrument(page 19)(2 hours)

Set up and run the real–time PCRinstrument(page 19)(2 hours)

▼ ▼

Analyze the results (page 26) Analyze the results (page 26)

Chapter 1 Product informationWorkflow 1


Prepare the sample and select theworkflow

Guidelines for total RNA input

• See Table 5 on page 10 for recommended RNA isolation kits.• The method used to isolate the total RNA must preserve the small RNA fraction.• To preserve endogenous control sequences in the total RNA, do not enrich for the

small RNA fraction. This may cause longer control transcripts (snoRNAs) to belost.

• Quantify the RNA to determine the appropriate workflow.

Select the workflow

This document describes two workflows:• With preamplification of the cDNA:

– Recommended for all samples– Important for samples with 1–350 ng of RNA– See Chapter 3, “Perform Megaplex™ Primer Pools with preamplification“

• Without preamplification of the cDNA:– Can be used for samples with 350–1000 ng of RNA– See Chapter 4, “Perform Megaplex™ Primer Pools without preamplification“

2


Perform Megaplex™ Primer Poolswith preamplification

■ Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■ Perform Megaplex™ reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

■ Perform the preamplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

■ Dilute the preamplification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

■ Perform PCR amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Procedural guidelines

• Use the TaqMan™ MicroRNA Reverse Transcription Kit and the Megaplex™ RTPrimers to synthesize single-stranded cDNA from total RNA samples.

• Perform one Megaplex™ RT reaction for each sample (one for each pool A and B)and optional no–template control, followed by one preamplification reaction persample or pool. This provides enough sample for one TaqMan™ Array Card.

• For a full MicroRNA profile, perform two Megaplex™ RT reactions (Pool A andPool B), two preamplification reactions (Pool A and Pool B), and two TaqMan™

Array Cards (A and B) per sample.• The RT reaction with downstream preamplification supports 1–1000 ng of input

total RNA.• For most tissues, 30 ng of total RNA produces a comprehensive MicroRNAprofile with preamplification.

• We recommend verifying the optimal quantity of input total RNA for yoursample type.

• Use 3 µL of RNA for each RT reaction, containing 1–350 ng of total RNA.

• Follow best-practices when preparing or performing PCR. For details, see “Bestpractices for PCR and RT-PCR experiments“ on page 42.

• Prepare the real-time PCR reactions in an area free of artificial templates andsiRNA transfections. High-copy-number templates can easily contaminate thereal-time PCR reactions.

• Before preparing a TaqMan™ Array Card, review Appendix B, “Detailedprocedures for preparation of a TaqMan™ Array Card“.

• Keep the card protected from light and stored as indicated until ready for use.Excessive exposure to light may affect the fluorescent probes of the dried-downassays in the card.

• Configure run documents according to the instructions provided in the real-timePCR instrument resource documents.

3

Guidelines forMegaplex™

reversetranscription withpreamplification

Guidelines forperforming real-time PCR


• Load each fill reservoir with 100 µL of sample-specific PCR reaction mix.– Each fill reservoir contains a single sample as determined by the card layout.– The 100–µL volume ensures adequate filling of each reaction well. Volumes

smaller than 100 µL result in insufficiently filled cards.• Equilibrate the card that is loaded with PCR reaction mix to room temperature

before loading into the real-time PCR instrument.• Run the card within 24 hours of sealing the card if TaqMan™ Universal Master

Mix II, no UNG is used.• Run the card within 72 hours of sealing the card if TaqMan™ Fast Advanced

Master Mix is used.• If the card is not run immediately, protect it from light and store at 2-8°C.

Perform Megaplex™ reverse transcription

Thaw the Megaplex™ RT Primers, TaqMan™ MicroRNA Reverse Transcription Kitcomponents, and MgCl2 on ice.

1. In an appropriately-sized microcentrifuge tube, prepare RT Reaction Mixaccording to the following table.

Component Volume(1 sample)

Volume(10 samples)[1]

Megaplex™ RT Primers (10X) 0.8 µL 9.00 µL

dNTPs with dTTP (100 mM) 0.2 µL 2.25 µL

MultiScribe™ Reverse Transcriptase(50 U/µL) 1.5 µL 16.88 µL

10X RT Buffer 0.8 µL 9.00 µL

MgCl2 (25 mM) 0.9 µL 10.12 µL

RNase Inhibitor (20 U/µL) 0.1 µL 1.12 µL

Nuclease-free water 0.2 µL 2.25 µL

Total RT Reaction Mix volume 4.5 µL 50.62 µL

[1] Includes 12.5% overage.

2. Invert to mix, then centrifuge briefly to collect the contents at the bottom of thetube.

3. Add 4.5 µL of the RT Reaction Mix to the wells of a reaction plate or to reactiontubes.

4. Add 3 µL of RNA or water (no template control) to each well of a reaction plateor to reaction tubes.3 µL of the RNA sample should contain 1-350 ng of total RNA.

5. Seal the reaction plate or tubes, invert to mix, then centrifuge briefly to collect thecontents at the bottom.

6. Incubate on ice for 5 minutes.

Chapter 3 Perform Megaplex™ Primer Pools with preamplificationPerform Megaplex™ reverse transcription3


7. Place the reaction plate or tubes into a thermal cycler, then incubate using astandard ramp speed and reaction volume of 7.5 µL.

Step Temperature Time Cycles

Reverse transcription

16°C 2 minutes

4042°C 1 minute

50°C 1 second

Stop reaction 85°C 5 minutes 1

Hold 4°C Hold untilstopped Hold

Proceed to the preamplification reaction or store the RT reaction product at–25°C to –15°C.

Perform the preamplification

Thaw the Megaplex™ PreAmp Primers on ice, invert to mix, then centrifuge briefly tocollect the contents at the bottom of the tube.

Thaw the RT reaction product on ice, if necessary.

1. Mix the TaqMan™ PreAmp Master Mix (2X) by swirling the bottle.

2. In an appropriately-sized microcentrifuge tube, prepare PreamplificationReaction Mix according to the following table.

Component Volume(1 sample) Volume (10 samples)[1]

TaqMan™ PreAmp Master Mix (2X) 12.5 µL 140.62 µL

Megaplex™ PreAmp Primers(10X) 2.5 µL 28.13 µL

Nuclease–free water 7.5 µL 84.37 µL

Total Preamplification Reaction Mixvolume 22.5 µL 253.12 µL


3. Invert to mix thoroughly, then centrifuge briefly to collect the contents at thebottom of the tube.

4. Add 2.5 µL of the RT reaction product to the wells of a reaction plate or toreaction tubes.

5. Add 22.5 µL of the Preamplification Reaction Mix to each well of a reaction plateor to reaction tubes.


7. Incubate the plate or tubes on ice for 5 minutes.

Chapter 3 Perform Megaplex™ Primer Pools with preamplificationPerform the preamplification 3


8. Place the reaction plate or tubes into a thermal cycler, then incubate using astandard ramp speed and the following settings.


Enzyme activation 95°C 10 minutes Hold

Anneal 55°C 2 minutes Hold

Extend 72°C 2 minutes Hold

Denature 95°C 15 seconds12[1]

Anneal/extend 60°C 4 minutes

Enzyme inactivation 99.9°C 10 minutes Hold

Hold 4°C Hold Hold

[1] The number of cycles can be optimized for extremely low input samples.

Proceed to the next section.

Dilute the preamplification reaction

1. Remove the reaction plate or tubes from the thermal cycler, then brieflycentrifuge to collect the contents at the bottom.

2. Add 75 µL of 0.1X TE pH 8.0 to each well or tube.

3. Seal the plate or tubes, invert to mix, then centrifuge briefly to collect thecontents at the bottom.

Place the diluted preamplification reaction product on ice and proceed directly to thereal-time PCR reactions (next section), or store at –25°C to –15°C for up to one week.

Chapter 3 Perform Megaplex™ Primer Pools with preamplificationDilute the preamplification reaction3


Perform PCR amplification

Thaw the RT reaction product on ice, then mix thoroughly by inverting the tube.

1. Mix the PCR Master Mix by swirling the bottle.

2. In an appropriately-sized microcentrifuge tube, prepare PCR Reaction Mixaccording to the following table.

Component Volume (1 card)[1]

PCR Master Mix 450 µL

Diluted preamplification product 9 µL

Nuclease–free water 441 µL

Total PCR Reaction Mix volume 900 µL



IMPORTANT! Before preparing a TaqMan™ Array Card, review Appendix B,“Detailed procedures for preparation of a TaqMan™ Array Card“.

1. Load each fill reservoir of the card with 100 µL of prepared PCR reaction mix.

2. Centrifuge, then seal the filled card.

See the appropriate instrument user guide for detailed instructions to program thethermal-cycling conditions or to run the card.

Note: The instrument must be configured with a block appropriate for a card.

1. Import the setup file (SDS in TXT format) into the software specific to yourinstrument.See “How to import setup files“ on page 38 for instructions to import setupfiles.

Note: The setup files are instrument-specific. See “Setup files for compatibleinstruments“ on page 38 for more information.

2. Set the properties for the run.

Property Setting

Block Array Card

Experiment type Comparative Ct (ΔΔCt)

Reagents or Chemistry TaqMan™ Reagents

Note: The default passive reference is set to ROX™ dye and should not bechanged.

Prepare PCRreactions

Prepare aTaqMan™ ArrayCard

Set up and run thereal–time PCRinstrument

Chapter 3 Perform Megaplex™ Primer Pools with preamplificationPerform PCR amplification 3


3. Select the properties.

• TaqMan™ Universal Master Mix II, no UNG—Standard• TaqMan™ Fast Advanced Master Mix—Fast

4. Set up the thermal protocol.

Note: Your thermal protocols might differ from the following tables in this userguide.

Table 10 TaqMan™ Fast Advanced Master Mix (ViiA™ 7 Real-Time PCR System,any compatible QuantStudio™ Real-Time PCR System)


Enzyme activation 92°C 10 minutes[1] 1

Denature 95°C 1 second40

Anneal / Extend 60°C 20 seconds

[1] To completely dissolve the assay on the card.

Table 11 TaqMan™ Fast Advanced Master Mix (7900HT Fast Real‑Time PCRSystem)






Table 12 TaqMan™ Universal Master Mix II, no UNG (ViiA™ 7 Real-Time PCRSystem, any compatible QuantStudio™ Real-Time PCR System)


Enzyme activation 95°C 10 minutes 1

Denature 95°C 15 seconds40


Table 13 TaqMan™ Universal Master Mix II, no UNG (7900HT Fast Real‑Time PCRSystem)


Enzyme activation 94.5°C 10 minutes 1

Denature 97.0°C 30 seconds40

Anneal / Extend 59.7°C 60 seconds

5. Confirm that the reaction volume is set to 1 µL.

6. Load the reaction card into the real-time PCR instrument.

7. Start the run.

Chapter 3 Perform Megaplex™ Primer Pools with preamplificationPerform PCR amplification3


Perform Megaplex™ Primer Poolswithout preamplification

■ Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

■ Perform Megaplex™ reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■ Perform PCR amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Procedural guidelines

• Use the TaqMan™ MicroRNA Reverse Transcription Kit and the Megaplex™ RTPrimers to synthesize single-stranded cDNA from total RNA samples.

• Perform one Megaplex™ RT reaction for each sample (one for each Pool A and B)and optional no–template control. This provides enough sample for oneTaqMan™ Array Card.

• For a full MicroRNA profile, perform two Megaplex™ RT reactions (Pool A andPool B) and optional no–template control. This provides enough sample for twoTaqMan™ Array Cards (A and B).

• The RT reaction supports 350–1000 ng of input total RNA.• For most tissues, 500 ng of total RNA produces a comprehensive MicroRNAprofile.

• We recommend verifying the optimal quantity of input total RNA for yoursample type.

• Use 3 µL of RNA for each RT reaction, containing 350–1000 ng of total RNA.

• Follow best-practices when preparing or performing PCR. For details, see “Bestpractices for PCR and RT-PCR experiments“ on page 42.

• Prepare the real-time PCR reactions in an area free of artificial templates andsiRNA transfections. High-copy-number templates can easily contaminate thereal-time PCR reactions.

• Before preparing a TaqMan™ Array Card, review Appendix B, “Detailedprocedures for preparation of a TaqMan™ Array Card“.


• Configure run documents according to the instructions provided in the real-timePCR instrument resource documents.

4

Guidelines forMegaplex™

reversetranscriptionwithoutpreamplification

Guidelines forperforming real-time PCR



smaller than 100 µL result in insufficiently filled cards.• Equilibrate the card that is loaded with PCR reaction mix to room temperature

before loading into the real-time PCR instrument.• Run the card within 24 hours of sealing the card if TaqMan™ Universal Master

Mix II, no UNG is used.• Run the card within 72 hours of sealing the card if TaqMan™ Fast Advanced

Master Mix is used.• If the card is not run immediately, protect it from light and store at 2-8°C.

Perform Megaplex™ reverse transcription

Thaw the Megaplex™ RT Primers, TaqMan™ MicroRNA Reverse Transcription Kitcomponents, and MgCl2 on ice.

1. In an appropriately-sized microcentrifuge tube, prepare RT Reaction Mixaccording to the following table.

Component Volume(1 sample)

Volume(10 samples)

[1]

Megaplex™ RT Primers (10X) 0.8 µL 9.00 µL

dNTPs with dTTP (100 mM) 0.2 µL 2.25 µL

MultiScribe™ Reverse Transcriptase(50 U/µL) 1.5 µL 16.88 µL

10X RT Buffer 0.8 µL 9.00 µL

MgCl2 (25 mM) 0.9 µL 10.12 µL

RNase Inhibitor (20 U/µL) 0.1 µL 1.12 µL

Nuclease-free Water 0.2 µL 2.25 µL

Total RT Reaction Mix volume 4.5 µL 50.62 µL



3. Add 4.5 µL of the RT Reaction Mix to the wells of a reaction plate or to reactiontubes.

4. Add 3 µL of RNA or water (no template control) to each well of a reaction plateor to reaction tubes.3 µL of the RNA sample should contain 350–1000 ng of total RNA.


Chapter 4 Perform Megaplex™ Primer Pools without preamplificationPerform Megaplex™ reverse transcription4


6. Incubate on ice for 5 minutes.

7. Place the reaction plate or tubes into a thermal cycler, then incubate using astandard ramp speed and reaction volume of 7.5 µL.


Reverse transcription

16°C 2 minutes

4042°C 1 minute

50°C 1 second

Stop reaction 85°C 5 minutes 1

Hold 4°C Hold untilstopped Hold

Proceed to the real–time PCR, or store the RT reaction product at –25°C to –15°C.

Perform PCR amplification

Thaw the RT reaction product on ice, then mix thoroughly by inverting the tube.

1. Mix the PCR Master Mix by swirling the bottle.

2. In an appropriately-sized microcentrifuge tube, prepare PCR Reaction Mixaccording to the following table.

Component Volume (1 card)[1]

PCR Master Mix 450 µL

cDNA 6 µL

Nuclease-free Water 444 µL

Total PCR Reaction Mix volume 900 µL



IMPORTANT! Before preparing a TaqMan™ Array Card, review Appendix B,“Detailed procedures for preparation of a TaqMan™ Array Card“.

1. Load each fill reservoir of the card with 100 µL of prepared PCR reaction mix.

2. Centrifuge, then seal the filled card.

Prepare PCRreactions

Prepare aTaqMan™ ArrayCard

Chapter 4 Perform Megaplex™ Primer Pools without preamplificationPerform PCR amplification 4


See the appropriate instrument user guide for detailed instructions to program thethermal-cycling conditions or to run the card.

Note: The instrument must be configured with a block appropriate for a card.

1. Import the setup file (SDS in TXT format) into the software specific to yourinstrument.See “How to import setup files“ on page 38 for instructions to import setupfiles.

Note: The setup files are instrument-specific. See “Setup files for compatibleinstruments“ on page 38 for more information.

2. Set the properties for the run.

Property Setting

Block Array Card

Experiment type Comparative Ct (ΔΔCt)

Reagents or Chemistry TaqMan™ Reagents

Note: The default passive reference is set to ROX™ dye and should not bechanged.

3. Select the properties.

• TaqMan™ Universal Master Mix II, no UNG—Standard• TaqMan™ Fast Advanced Master Mix—Fast

4. Set up the thermal protocol.

Note: Your thermal protocols might differ from the following tables in this userguide.

Table 14 TaqMan™ Fast Advanced Master Mix (ViiA™ 7 Real-Time PCR System,any compatible QuantStudio™ Real-Time PCR System)






Table 15 TaqMan™ Fast Advanced Master Mix (7900HT Fast Real‑Time PCRSystem)






Set up and run thereal–time PCRinstrument

Chapter 4 Perform Megaplex™ Primer Pools without preamplificationPerform PCR amplification4


Table 16 TaqMan™ Universal Master Mix II, no UNG (ViiA™ 7 Real-Time PCRSystem, any compatible QuantStudio™ Real-Time PCR System)


Enzyme activation 95°C 10 minutes 1

Denature 95°C 15 seconds40


Table 17 TaqMan™ Universal Master Mix II, no UNG (7900HT Fast Real‑Time PCRSystem)


Enzyme activation 94.5°C 10 minutes 1

Denature 97.0°C 30 seconds40

Anneal / Extend 59.7°C 60 seconds

5. Confirm that the reaction volume is set to 1 µL.

6. Load the reaction card into the real-time PCR instrument.

7. Start the run.

Chapter 4 Perform Megaplex™ Primer Pools without preamplificationPerform PCR amplification 4


Analyze data

Analyze the results

For detailed information about data analysis, see the appropriate documentation foryour instrument.

Use the relative quantification (ΔΔCt) method to analyze results.

A cutoff of 32 is recommended. If pre-amplification is used, the cutoff can be set to 29or 30 to reduce the number of false positives.

The general guidelines for analysis include:• View the amplification plot; then, if needed:

– Adjust the baseline and threshold values.– Remove outliers from the analysis.

• In the well table or results table, view the Ct values for each well and for eachreplicate group.

Perform additional data analysis using any of the following software:

Software Resource

Relative Quantification application thermofisher.com/cloud

ExpressionSuite™ Software[1] thermofisher.com/expressionsuite

[1] Can automatically define the baseline.

For more information about real-time PCR, see “Set up and run the real–time PCRinstrument“ on page 19 or go to thermofisher.com/qpcreducation.

Ct (Cq) values can be generated using the relative threshold algorithm (Crt).

Use the relative threshold algorithm in your software. The relative thresholdalgorithm is available on the following instruments:

• QuantStudio™ Real-Time PCR Instruments• ViiA™ 7 instrument

If your software does not have the relative threshold algorithm, use the RelativeQuantification application that is available on the Connect.

Note: The Ct algorithm is recommended on the 7900HT Fast Real-Time PCRInstrument.

See Introduction to Gene Expression Getting Started Guide (Pub. No. 4454239) forinformation about Ct and Crt.

5




http://www.thermofisher.com/qpcreducation

Table 18 Algorithm recommendations for TaqMan™ Array Cards

Algorithm Recommendation

Relative threshold (Crt)

Recommended for the following instruments:

• QuantStudio™ Real-Time PCR Instruments

• ViiA™ 7 instrument

Can correct a variable baseline, which might be due todried-down assays on the card being reconstituted atdifferent rates.

Threshold (Ct)Optional if used for analysis of established protocols.

Recommended for 7900HT Fast Real-Time PCRInstrument.

The relative threshold algorithm is available in the Relative Quantification applicationon Connect (thermofisher.com/connect).

Each TaqMan™ Array Card of the two-card set (either human or rodent) containsidentical candidate endogenous control assays that provide a variety of options fornormalization.

Endogeneous control Species Notes

Mammalian U6 Human, mouse, andrat

Repeated 4x on card enablingmonitoring of assay reproducibility

RNU48 Human Small nucleolar RNA

RNU44 Human Small nucleolar RNA

snoRNA135 Mouse Small nucleolar RNA

snoRNA202 Mouse Small nucleolar RNA

U87 Rat Small nucleolar RNA

Y1 Rat Small non-coding RNA

ath-miR159a ArabidopsisNot detectable in mammalian speciesFor use as a negative or processingcontrol

Algorithms fordata analysis

Datanormalization

Chapter 5 Analyze dataAnalyze the results 5


https://www.thermofisher.com/connect

Megaplex™ RT Primers and Megaplex™ PreAmp Primers are highly multiplexed poolsdesigned to significantly streamline the workflow when profiling many miRNAtargets in a single experiment. When combined in a large multiplex pool, a smallsubset of assays exhibit lower no-template-control (NTC Ct values <35) values thanwhen run individually. This is not an assay design issue. In the interest of providingthe broadest coverage possible, customers have requested that we include theseassays on the profiling array, but with the expectation that they are to be consideredsemi-quantitative. As a result, fold-change measurements using these assays may beless than the true value. To aid data interpretation, the identity of these assays isprovided in the Megaplex™ Assay Performance File. If accurate fold-changemeasurements for these assays is required, we recommend that changes detectedusing the Megaplex™ assay workflow be validated using the corresponding individualTaqMan™ MicroRNA Assay.

The Megaplex™ Assay Performance File is found in the Product literature section ofeach TaqMan™ Array Card page at thermofisher.com.

File TaqMan™ Array Card Cat. No.

Megaplex™ Assay Performance File Human Card A 4398965

Megaplex™ Assay Performance File Human Card B 4444910

Megaplex™ Assay Performance File Rodent Card A 4398967

Megaplex™ Assay Performance File Rodent Card B 4444899

Megaplex™ assayperformance

Megaplex™ AssayPerformance File

Chapter 5 Analyze dataAnalyze the results5


http://www.thermofisher.com

Troubleshooting and FAQs

Visit our online FAQ database for tips and tricks for conducting your experiment,troubleshooting information, and FAQs. The online FAQ database is frequentlyupdated to ensure accurate and thorough content.

• For troubleshooting information and FAQs for this product: thermofisher.com/megaplexpoolsformicroRNAfaqs

• To browse the database and search using keywords: thermofisher.com/faqs

A


http://www.thermofisher.com/megaplexpoolsformicroRNAfaqs

http://www.thermofisher.com/megaplexpoolsformicroRNAfaqs

https://www.thermofisher.com/faqs

Detailed procedures for preparationof a TaqMan™ Array Card

■ Guidelines for preparation of a card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

■ TaqMan™ Array Card diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

■ Load the PCR reaction mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

■ Centrifuge the card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

■ Seal the card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Guidelines for preparation of a card


• Before removing the card from its packaging:– Prepare each sample-specific PCR reaction mix.– Allow the card to reach room temperature.


smaller than 100 µL result in insufficiently filled cards.• Do not allow the micropipette tip to contact the coated foil beneath the fill port.

B


• Load the card with PCR reaction mix before centrifuging the card.During centrifugation, the PCR reaction mix resuspends the dried-down assaysin each well of the card. Adding sample after centrifuging disrupts the assaylayout of the card.

• Store the sealed card, protected from light, at 2–8°C. Run the card within the timeallowed by the Master Mix. For procedures with preamplification see “Guidelinesfor performing real-time PCR“ on page 15. For procedures withoutpreamplification see “Guidelines for performing real-time PCR“ on page 15.

• If the card is not run immediately, protect it from light and store at 2-8°C.

TaqMan™ Array Card diagram

A TaqMan™ Array Card includes 8 fill reservoirs and 384 reaction wells.

GR2156a Microfluidic Card Small file (compared to 2156)

1

2

3

4

5

1 Fill reservoirs (8 total)2 Fill reservoir strip3 Array card carrier

4 Array card barcode5 Reaction well (384 total)

1

2

2

1 Foil2 Array card feet

The fill reservoir includes a fill port and a vent port. Use the fill port to load PCRreaction mix into the card.

Appendix B Detailed procedures for preparation of a TaqMan™ Array CardTaqMan™ Array Card diagram B


GR2158Microfluidic CardCard Ports detail

21

1 Fill port2 Vent port

Load the PCR reaction mix

Before removing the card from its packaging:• Prepare each sample-specific PCR reaction mix.• Allow the card to reach room temperature.

1. Carefully remove the card from its packaging.

2. Place the card on the benchtop with its foil-side down.

3. Load 100 µL of the sample-specific PCR reaction mix into a micropipette.

4. Hold the micropipette in an angledposition, then place the tip into a fillport of the card.

5. Slowly dispense the entire volume ofreaction mix so that it sweeps in andaround the fill reservoir toward thevent port.

Centrifuge the card

1. Load the cards into the centrifuge buckets.a. Place the bucket on the benchtop with its label facing the front of the bench.

b. Insert the cards into the card holder, ensuring that:• The fill reservoirs extend upwards out of the card holder.• The reaction wells face the label-side of the card holder.

c. Insert blank balance cards into any empty positions of the card holder. Allthree positions in the card holder must be filled.

Appendix B Detailed procedures for preparation of a TaqMan™ Array CardLoad the PCR reaction mixB


d. Place the loaded card holder into the bucket so that the card holder labelfaces the front of the bucket.

GR2155Microfluidic CardPlates into clip ►

GR2160 Microfluidic Card Clip into bucket

2. Configure the centrifuge using its front-panel controls.a. Set the bucket type to 15679.

b. Set the following parameters according to the control panel type.

Parameter EASYSet(touchpad-operated)

QUIKSet(knob-operated)

Increasing ramp rate 9 3

Decreasing ramp rate 9 —

Rotational speed 1,200 rpm (331 × g) 1,200 rpm

Centrifugation time [1] 1 minute 1 minute

[1] You will centrifuge the cards twice, each time for 1 minute (see step 4).

IMPORTANT! A speed that is set too high can deform the card.

3. Load the buckets into the centrifuge.

a. Press on the centrifugecontrol panel to open thecentrifuge cover.

b. Place each loaded bucket ontoan open rotor arm of thecentrifuge.Ensure that each bucket canswing easily within its slottedposition on the rotor arm.

c. If there are empty rotor arms,prepare buckets with blankbalance cards as described in step 1. Place the balance bucketsonto the rotor arms.The rotor must be evenly loadedand opposing buckets must hold the same weight.

GR2161 Microfluidic Card Centrifuge half full

Centrifuge is properly loaded and balanced.

Appendix B Detailed procedures for preparation of a TaqMan™ Array CardCentrifuge the card B


d. Close the centrifuge cover.

4. Run the centrifuge.a. Press .

The centrifuge will start, then automatically stop after 1 minute.

b. Repeat substep 4a so that the cards are centrifuged for a total of two,consecutive, 1-minute centrifugations.

IMPORTANT! Do not centrifuge the cards continuously for 2 minutes. Theramping up in speed during the two, consecutive 1‑minute centrifugations isimportant for proper filling.

5. Remove the cards from the centrifuge.a. Press .

b. Remove the buckets from the centrifuge, then remove the card holders fromthe buckets.

c. Remove each card from the card holder by lifting it gently by the cardcarrier sides.

6. Examine the cards for proper filling.When properly filled, the remaining volumes of PCR reaction mix are consistentreservoir to reservoir. If the card is not properly filled, see Appendix A,“Troubleshooting and FAQs“.

Consistent filling Inconsistent filling

GR2157Microfluidic CardCard structure

GR2162Microfluidic CardBad fill 1

GR2163Microfluidic CardBad fill 2

Seal the card

The TaqMan™ Array Card Sealer isolates the wells of an array card after it is loadedwith PCR reaction mix and centrifuged. The sealer uses a precision stylus assembly(under the carriage) to seal the main fluid distribution channels of the array card.

Note: In some documents, the TaqMan™ Array Card Sealer is referred to as a StylusStaker.

1. Position the TaqMan™ Array CardSealer and its carriage beforeinserting a card.

a. Place the sealer on a benchtopthat is approximatelywaist-high so that the carriagecan be easily maneuvered.

GR2172aMicroFluidic CardSealer with card

3

2

1

1 Carriage

Appendix B Detailed procedures for preparation of a TaqMan™ Array CardSeal the cardB


b. Position the sealer with thecarriage starting positiontoward the front of the bench.Ensure that the engraved arrows on the sealer point away from you.

c. Ensure that the carriage for the sealer is at the starting position.

IMPORTANT! Do not insert a filled card into the sealer if the carriage is notin its starting position. The card will be irreparably damaged if the carriageis moved backwards across the card towards the starting position.

2. Insert a filled, centrifuged card into the sealer.a. Hold the card with its foil-side up.

b. Orient the card over the sealer with the fill reservoirs of the card toward theending position.

c. Align the rear pin grooves of the card to the alignment pins of the sealer.

GR2174a MicroFluidic Card Spring Plate

2

2

1

1 Alignment pins2 Spring clips

GR2172aMicroFluidic CardSealer with card

d. Gently place the card on top of the sealer.

e. Gently push the card until it isseated securely in the sealer.When properly seated, the foilsurface of the card is level withthe base of the sealer and thespring clips hold the card securelyin place.

2 Carriage starting position3 Carriage ending position

GR2174aMicroFluidic CardSpring Plate

Appendix B Detailed procedures for preparation of a TaqMan™ Array CardSeal the card B


3. Slowly and steadily push the carriage across the sealer in the direction of theengraved arrows.Push the carriage across the entire length of the card until the carriage meets themechanical stops at the ending position.

GR2172MicroFluidic CardSealer with plate ► GR2176

MicroFluidic CardSealer position end

IMPORTANT!· Do not use excessive force or speed when pushing the carriage across the card.· Do not move the carriage back across the card. Leave the carriage at the

ending position while removing the card from the sealer.

4. Remove the sealed card from the sealer by grasping the sides of the card andlifting it off.Use the thumb slot in the middle of the sealer to access the card.

5. Examine the card for proper sealing.

Note: When properly sealed, the indentations from the sealer carriage align withthe main channels of the card.If the indentations do not align or if the foil is damaged, do not use the card.

6. Use scissors to trim the fill reservoir strip from the card. Use the edge of the cardcarrier as a guide.

GR2168Microfluidic CardTrim Edge

IMPORTANT! Completely remove the fill reservoir strip. Any remaining plasticthat extends beyond the card edge can prevent the card from seating properly onthe sample block and can affect amplification.

Correct trim Incorrect trim

The card is now ready to run on the instrument.

Appendix B Detailed procedures for preparation of a TaqMan™ Array CardSeal the cardB


Store the sealed card, protected from light, at 2–8°C. The length of time the card canbe stored depends on the Master Mix. See “Perform the preamplification“ on page 17for procedures with preamplification, and “Prepare PCR reactions“ on page 23 forprocedures without preamplification.

Appendix B Detailed procedures for preparation of a TaqMan™ Array CardSeal the card B


Supplemental information

How to import setup files

Instrument Navigation

QuantStudio™ 7 Flex System

In the menu bar during setup, select File4Import PlateSetup.QuantStudio™ 12K Flex Real–Time PCR System


7900HT Fast Real‑Time PCR System In the menu bar, select File4New Plate Wizard, thenfollow the prompts.

Setup files for compatible instruments

Instrument Setup file

QuantStudio™ 7 Flex System

SDS_order number.txtQuantStudio™ 12K Flex Real–Time PCR System


7900HT Fast Real‑Time PCR System

C


Megaplex™ Primer Pools chemistry overview

RNA samples are reverse transcribed to cDNA. The reverse transcription reactionuses specific MicroRNA primers and reagents from the Megaplex™ Primer Pools andthe TaqMan™ MicroRNA Reverse Transcription Kit.

First cDNA strand

Looped RT primer

Micro RNA

Reverse transcriptase

DNA polymerase

RT

P

3'P

5'

3'5'3'

3'5'RT

3'5'

5'3'

Figure 1 Reverse transcription reaction with looped primer

cDNA templates are uniformly preamplified with the Megaplex™ PreAmp Primersand TaqMan™ PreAmp Master Mix. The resulting preamplified product is amplifiedwith real–time PCR.

cDNA template

cDNA template

cDNA template

DNA polymerase

Reverse primer

Forward primer

P

Reversetranscription

Preamplification

Appendix C Supplemental informationMegaplex™ Primer Pools chemistry overview C


3' 5'5' 3'

5' 3'P

3' 5'P

5'3'5' 3'

5'3'5' 3'

3'5'P

5'3'P

Figure 2 Preamplification reaction

TaqMan™ MGB probes contain:• A reporter dye (for example, FAM™ dye) 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™ MicroRNA Assays. The sequence regions arenot 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 cDNA synthesisproduct.

cDNA template

Target region of cDNA template

Forward primer

Reverse primer

Probe

Reporter dye

Fluorescing reporter dye

Non-fluorescent quencher

R

R

NFQ

TaqMan™ MGBprobes

About the 5'nuclease assay

Appendix C Supplemental informationMegaplex™ Primer Pools chemistry overviewC


Minor groove binder probe

MGB

3' 5'5' 3'

Figure 3 cDNA synthesis product or preamplified cDNA synthesis product

During the PCR, the forward and reverse primers anneal to complementary sequencesalong the denatured cDNA template strands.

The TaqMan™ MGB probe anneals specifically to a complementary sequence betweenthe forward and reverse primer sites. When the probe is intact, the proximity of thereporter dye and quencher dye suppresses the reporter fluorescence, primarily byFörster-type energy transfer.

3' 5'

5' 3'

R NFQ MGB

Figure 4 Annealing of probes and primers to cDNA strands or preamplified cDNA strands

During polymerization, the DNA polymerase only cleaves 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.

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.

Figure 5 Initial polymerization and cleavage of reporter dye

Polymerization of the strand continues, but because the 3' end of the probe is blocked,no extension of the probe occurs during PCR.

Appendix C Supplemental informationMegaplex™ Primer Pools chemistry overview C


3' 5'

5' 3'

R

Figure 6 Completion of polymerization

Best practices for PCR and RT-PCR experiments

• Wear clean gloves and a clean lab coat.– Do not wear the same gloves and lab coat that you have previously used

when 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 C Supplemental informationBest practices for PCR and RT-PCR experimentsC


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, and so on). To obtain SDSs, see the “Documentationand Support” section in this document.

D


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 sufficient ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer 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 needed) 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.

WARNING! HAZARDOUS WASTE (from instruments). Waste produced bythe instrument is potentially hazardous. Follow the guidelines noted in thepreceding General Chemical Handling warning.

WARNING! 4L Reagent and Waste Bottle Safety. Four-liter reagent and wastebottles can crack and leak. Each 4-liter bottle should be secured in a low-densitypolyethylene safety container with the cover fastened and the handles locked inthe upright position.

Appendix D SafetyChemical safetyD


Biological hazard safety

WARNING! Potential Biohazard. Depending on the samples used on thisinstrument, the surface may be considered a biohazard. Use appropriatedecontamination methods when working with biohazards.

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:https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.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 D SafetyBiological hazard safety D


https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf

https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf

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

Documentation and support

Related documentation

Document Pub. No.

Megaplex™ Primer Pools for MicroRNA expression analysis Quick Reference 4399813

Megaplex™ Primer Pools Product Information Sheet 4401697

Understanding Your Shipment(for detailed information about the Assay Information File) MAN0017153

Applied Biosystems™ Relative Quantitation Analysis Module User Guide MAN0014820

QuantStudio™ 7 Flex Real-Time PCR System

QuantStudio™ 6 and 7 Flex Real-Time PCR Systems Maintenance and Administration Guide 4489821

QuantStudio™ 6 and 7 Flex Real-Time PCR System Software Getting Started Guide 4489822

QuantStudio™ 12K Flex Real–Time PCR System

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


Applied Biosystems™ ViiA™ 7 Real-Time PCR System User Guide: Calibration, Maintenance,Networking, and Security 4442661

Applied Biosystems™ ViiA™ 7 Real-Time PCR System Getting Started Guide 4441434

7900HT Real-Time PCR System

Applied Biosystems™ 7900HT Fast Real-Time PCR System Maintenance and TroubleshootingGuide 4365542

Applied Biosystems™ 7900HT Fast Real-Time PCR System User Bulletin 4352533


Customer and technical support

Visit 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 affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you haveany questions, please contact Life Technologies 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/support

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