TaqMan Gene Expression Assay Protocol

TaqMan® Gene Expression Assays
Protocol
For Research Use Only. Not for use in diagnostic procedures.
Information in this document is subject to change without notice.
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TRADEMARKS
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© 2010 Life Technologies Corporation. All rights reserved.
Part Number 4333458 Rev. N

11/2010
Contents
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Safety alert words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
PROTOCOL TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Purpose of the product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Assay formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Storage and dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Materials and equipment not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Endogenous control assay(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Materials for reverse transcription and PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Compatible real-time instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Prepare the cDNA sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Isolate total RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Perform reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Evaluate the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
(Optional) Store the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Prepare the reaction mix and load the plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Thaw and mix the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Calculate the number of reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Prepare the PCR reaction mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Load the plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Run the real-time PCR reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Create the plate document/experiment and run the plate . . . . . . . . . . . . . . . . . . . . . . . 18
Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Resources for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Tools for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
TaqMan® Gene Expression Assays Protocol 3

Contents
APPENDIX A How to Order TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . 31

Step 1: Search for an assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Search for TaqMan® Gene Expression Assays or Non-coding RNA Assays . . . . . . . . . 32
Search for TaqMan® Gene Expression Assays only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
About TaqMan® Gene Expression Assay IDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Step 2: Order the assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Order TaqMan® Gene Expression Assays and TaqMan® Non-coding RNA Assays . . . 35
Order Custom TaqMan® Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Reorder a Custom TaqMan® Gene Expression Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Step 3: Order a candidate endogenous control assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Assays for normalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Step 4: Select a chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Select standard or Fast chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Select 1- or 2-step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Step 5: Order materials and equipment not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Related reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Reaction plates and accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Related gene expression assays and arrays products . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
APPENDIX B Good PCR Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Prevent contamination and nonspecific amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
AmpErase® UNG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
PCR good laboratory practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
APPENDIX C Duplex Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Duplex reactions using TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Setting up a Duplex Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
APPENDIX D Background Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

About TaqMan® chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
About the probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
About the 5′ nuclease assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
About the assay information file (AIF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
AIF formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
AIF field descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
APPENDIX E Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
General chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Chemical waste safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
General safety alerts for all chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4 TaqMan® Gene Expression Assays Protocol

Contents
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Contents

About This Guide
Purpose
This TaqMan® Gene Expression Assays Protocol provides instructions for performing
real-time reverse transcription-PCR (real-time RT-PCR) using TaqMan Gene
Expression Assays and TaqMan Non-coding RNA Assays.
Safety information
Note: For general safety information, see this section and Appendix E, “Safety”
on page 59. When a hazard symbol and hazard type appear by an instrument
hazard, see the “Safety” Appendix for the complete alert on the instrument.
Safety alert words

Four safety alert words appear in Applied Biosystems user documentation at points in
the document where you need to be aware of relevant hazards. Each alert word—
IMPORTANT, CAUTION, WARNING, DANGER—implies a particular level of
observation or action, as defined below:
IMPORTANT! – Indicates information that is necessary for proper instrument
operation, accurate chemistry kit use, or safe use of a chemical.
CAUTION! – Indicates a potentially hazardous situation that, if not avoided,

may result in minor or moderate injury. It may also be used to alert against
unsafe practices.
WARNING! – Indicates a potentially hazardous situation that, if not avoided,

could result in death or serious injury.
DANGER! – Indicates an imminently hazardous situation that, if not avoided,

will result in death or serious injury. This signal word is to be limited to the most
extreme situations.

About This Guide
Safety information
SDSs
The SDSs for any chemicals supplied by Applied Biosystems or Ambion are available
to you free 24 hours a day. For instructions on obtaining SDSs, see “SDSs” on page 61.
IMPORTANT! For the SDSs of chemicals not distributed by Applied Biosystems
or Ambion contact the chemical manufacturer.

PROTOCOL
Product information
Purpose of the product
Applied Biosystems offers comprehensive collections of predesigned, preformulated
primer and probe sets that help researchers perform quantitative gene expression
studies on a variety of species.
• TaqMan® Gene Expression Assays – Target protein-coding transcripts from a
variety of species, including human, mouse, rat, Arabidopsis, C. elegans, and
Drosophila. See Table 6 on page 33 for a complete list of species.
• TaqMan® Non-coding RNA Assays – Target long non-coding RNA (ncRNA) in
human, mouse, and rat species. These assays are designed to ncRNAs that are
>60 nt in length.
This protocol provides instructions for real-time reverse transcription-PCR (real-time
RT-PCR) using TaqMan Gene Expression Assays and TaqMan Non-coding RNA
Assays. Both assays are compatible with the same instruments and master mixes, and
real-time RT-PCR is performed using the same procedure. Unless explicitly stated
otherwise, the term “TaqMan Gene Expression Assays” is used throughout this guide
to mean either assay type.
For information about TaqMan assay reactions, refer to “About TaqMan® chemistry”
on page 51.
Assay formulations
TaqMan Gene Expression Assays are available as:
• Inventoried Assays – Predesigned real-time PCR assays that are previously
manufactured and immediately available at the time you submit an order.
• Made-to-Order Assays – Predesigned real-time PCR assays that are
manufactured at the time you submit an order.
• Custom Assays – Custom assays designed for you to target any sequence within
a gene, either across exon boundaries or within an exon. Submit a target sequence
for any organism and Applied Biosystems sends you a ready-to-use gene
expression assay with optimized primers and probe.
• TaqMan® Endogenous Controls – A collection of predesigned assays for
candidate control genes used to normalize for differences in sample RNA added
to a reaction. A number of candidate endogenous control genes are available for
use with TaqMan® Gene Expression Assays or TaqMan Non-coding RNA Assays.
For more information on selecting endogenous controls, see “Step 3: Order a
candidate endogenous control assay” on page 36.
TaqMan Non-coding RNA Assays are available as Made-to-Order Assays.

Product information
Kit contents
TaqMan Gene Expression Assays and TaqMan Non-coding RNA Assays include:
• One tube for each assay that is ordered. The tube contains:
– Two unlabeled primers (1✕ final concentration is 900 nM per primer;
20✕ stock concentration is 18 µM per primer)
– One 6-FAM™ dye-labeled TaqMan® MGB probe (1✕ final concentration is
250 nM; 20✕ stock concentration is 5 µM)
– (TaqMan Gene Expression Assays only) One 6-VIC® dye-labeled TaqMan
MGB probe (1X concentration is 250nM; 20X stock concentration is 5 µM);
also available as primer limited (1X final concentration is 150 nM per primer;
20X stock concentration is 3 µM per primer)
Note: The assay ID that appears on the tube of each TaqMan® Gene
Expression Assay is a unique, alphanumeric string that identifies the assay
and encodes basic descriptive information. See “About TaqMan® Gene
Expression Assay IDs” on page 33 for more information.
• A data sheet containing information about the assay.
• An Information CD that includes the following files:
– Assay information file (AIF)
– TaqMan® Gene Expression Assays Protocol (PN 4333458)
– TaqMan® Gene Expression Assays Quick Reference Card (PN 4401212)
– Understanding Your Shipment, included with TaqMan Non-coding RNA
Assays and certain TaqMan Gene Expression Assays
Table 1 TaqMan® Assay formulations
Product Type 20-µL rxns. Part number Availability
TaqMan® Gene Extra 75 4453320 Inventoried (20X)

Expression Assays Small
(FAM™ dye-labeled
Extra 75 4448892 Made-to-Order (20✕)
MGB probe)
Small
Small 250 4331182 Inventoried (20✕)
Small 360 4351372 Made-to-Order (20✕)
Medium 750 4351370
Large 2900 4351368 Made-to-Order (60X)
TaqMan® Gene Small 360 4448489 Made-to-Order (20✕)
Expression Assays
Medium 750 4448490
(VIC® dye-labeled
MGB probe) Large 2900 4448491 Made-to-Order (60✕)
TaqMan® Gene Small 360 4448484 Made-to-Order (20✕)
Expression Assays
Medium 750 4448485
Primer-Limited
(VIC® dye-labeled Large 2900 4448486 Made-to-Order (60✕)
MGB probe)

Product information
Product Type 20-µL rxns. Part number Availability
TaqMan® Non- Small 360 4426961 Made-to-Order (20✕)

coding RNA Assay
Medium 750 4426962
Large 2900 4426963 Made-to-Order (60✕)
Custom Plus Small 360 4441114 Custom (20✕)
TaqMan® RNA Assay
Medium 750 4441117
Large 2900 4441118 Custom (60✕)
Custom TaqMan® Small 360 4331348 Custom (20✕)
Gene Expression
Medium 750 4332078
Assays
Large 2900 4332079 Custom (60✕)
TaqMan® Small 125 – ‡ Inventoried (20✕)
Endogenous
Medium 500
Controls (20✕):
• Non-primer Large 2500
limited, FAM™
dye-labeled MGB
probe
• Primer limited,
VIC® dye-labeled
MGB probe
• Primer limited,
VIC® dye-labeled
probe
• TAMRA™ dye-
quenched probe
‡ See “Step 3: Order a candidate endogenous control assay” on page 36.
Ordering an assay For details on how to order an assay, refer to the TaqMan® Gene Expression Assays
products page at www.allgenes.com or Appendix A, “How to Order TaqMan® Gene
Expression Assays” on page 31.
Storage and dilution

• Store TaqMan Gene Expression Assay and TaqMan Non-coding RNA Assay
products at –15 to –25 °C and keep them protected from light.
• To minimize freeze-thaw cycles, consider diluting 60✕ assays to 20✕ working
stocks and dividing the solutions into smaller aliquots.

Materials and equipment not included

Endogenous control assay(s)
TaqMan® Endogenous Controls are a collection of predesigned assays for candidate
control genes, used to normalize for differences in sample RNA added to a reaction.
For a list of endogenous control assays, refer to “Step 3: Order a candidate endogenous
control assay” on page 36. For more information on selecting an endogenous control,
refer to the Application Note: Using TaqMan® Endogenous Control Assays to Select an
Endogenous Control for Experimental Studies (Stock Number 127AP08-01), available at:
www.appliedbiosystems.com
Materials for reverse transcription and PCR

Obtain the following materials for the reverse transcription and PCR (see “Step 5:
Order materials and equipment not included” on page 41 for a complete list of
materials). Unless otherwise indicated, all materials are available from major
laboratory suppliers (MLS).
Table 2 Required materials and equipment
✔ Material Source
Reverse transcription reagents Applied Biosystems

(See Table 11 on page 41)
PCR reagents
Thermal cycler (or real-time PCR instrument) Applied Biosystems
Real-time PCR instrument
Reaction plates and accessories for your real-time PCR Applied Biosystems
instrument (See Table 12 on page 42)
Centrifuge (with plate adapter) MLS
Disposable gloves MLS
Microcentrifuge MLS
Pipette tips, aerosol-resistant MLS
Pipettors (positive/air-displacement or multichannel) MLS
Polypropylene tubes (various sizes) MLS
Vortexer MLS
Nuclease-free water (no diethyl pyrocarbonate [DEPC]) MLS

Compatible real-time instruments

TaqMan Gene Expression Assays and TaqMan Non-coding RNA Assays can be used
with the Applied Biosystems:
• 7300 Real-Time PCR System
• 7500 Real-Time PCR System
• 7500 Fast Real-Time PCR System
• 7900HT Real-Time PCR System
• StepOne™ Real-Time PCR System
• StepOnePlus™ Real-Time PCR System
• ViiA™ 7 Real-Time PCR System

Workflow
Workflow
Prepare the cDNA sample
Prepare the total RNA
Perform reverse transcription

High Capacity RNA-to-cDNA Kit
Evaluate the cDNA GeneAm

PCR Systemp ®
9700
Thermal cycler or
real-time PCR instrument
Prepare the reaction mix and load the plate
Prepare the PCR reaction mix
TaqMan® Gene Expression

Load the plate Master Mix
1 2 3 4 5 6 7 8 9 10 11 12
A
48-, 96-, or 384-well

reaction plate
Run the real-time PCR reaction
Create the plate document/experiment

System software and information CD
Run the plate

or or
Real-time PCR instrument
Analyze the results
Refer to the user guide for your real-time PCR

instrument
Amplification plot


Isolate total RNA
Before running the TaqMan Gene Expression Assays, isolate total RNA to use as a
template for synthesis of single-stranded cDNA. For optimal performance, Applied
Biosystems recommends using an Ambion® RNA isolation kit. Go to
www4.appliedbiosystems.com, select RNA Isolation Which RNA Isolation Kit to
Choose? to view a list of kits.
Applied Biosystems recommends using total RNA that is:
• Between 0.002 and 0.2 µg/µL
• Less than 0.005% of genomic DNA by weight
IMPORTANT! Assays designed to a single exon (assay IDs with _s and _g
suffixes) will detect genomic DNA. When using these types of assays, if
your RNA purification method does not include DNase treatment, treat the
purified RNA with the Ambion® TURBO DNA-free™ Kit (recommended;
PN AM1907) using the standard protocol.
• Dissolved in a PCR-compatible buffer
• Free of RNase activity
• Free of inhibitors of reverse transcription and PCR
• Nondenatured
IMPORTANT! Denaturation of the RNA is not necessary and may reduce
the yield of cDNA for some gene targets.
Perform reverse transcription

Applied Biosystems recommends using one of the following kits to obtain cDNA from
RNA samples.
• High Capacity RNA-to-cDNA Kit (PN 4387406)
• High Capacity cDNA Reverse Transcription Kit (PN 4368813, 4374966)
Note: Use the same reverse transcription procedure for all samples in an
experimental study. See Table 11 on page 41 for a list of compatible reverse
transcription kits.
Evaluate the cDNA

Applied Biosystems recommends that you use:
• 1 to 100 ng of cDNA per 20-µL amplification reaction (PCR)
• The same amount of cDNA in each reaction

DNA quantitation Applied Biosystems recommends that you use:

methods • TaqMan® RNase P Detection Reagents (recommended; PN 4316831). These
reagents enable quantitation of cDNA that is able to function as a template in
PCR. Refer to Creating Standard Curves with Genomic DNA or Plasmid DNA
Templates for Use in Quantitative PCR (search for PN 4371090 at
www.appliedbiosystems.com).
or
• UV absorbance (A260/A280) measurements.
(Optional) Store the cDNA

If you do not proceed immediately to PCR amplification, store all cDNA samples at
−15 to −25 °C. To minimize freeze-thaw cycles, store the cDNA in smaller aliquots.

Thaw and mix the reagents
1. Thaw on ice, completely resuspend by gently vortexing, then briefly centrifuge to
bring liquid to the bottom of the tube:
• TaqMan Gene Expression Assays (20✕)
• cDNA samples
2. Mix the master mix reagent by gently swirling the bottle (see Table 11 on page 41
for a list of compatible master mixes available from Applied Biosystems).
Calculate the number of reactions

Calculate the number of reactions that you need for each assay. Applied Biosystems
recommends performing four replicates of each reaction. Be sure to include on each
plate:
• A TaqMan Gene Expression Assay for each cDNA sample
• Endogenous control assays
• (Optional) No template controls (NTCs) for each gene expression assay on the
plate

Prepare the PCR reaction mix

For duplex reactions using VIC dye-labeled, primer-limited assays, see Appendix C,
“Duplex Reactions” on page 49.
1. For each sample (to be run in quadruplicate), pipet the following into a nuclease-
free 1.5-mL microcentrifuge tube:
Volume per 20-µL reaction (µL)

PCR reaction mix component
Single Four
reaction replicates#
20✕ TaqMan® Gene Expression Assay 1.0 5.0

2✕ TaqMan® Gene Expression Master Mix‡ 10.0 50.0
cDNA template (1 to 100 ng)§ 4.0 20.0
RNase-free water 5.0 25.0
‡ (Optional) Use TaqMan® Fast Advanced Master Mix or TaqMan® Universal Master Mix. If you add
AmpErase® UNG (uracil-N-glycosylase), the final concentration must be
0.01 U/µL. Reduce the volume of water in the PCR reaction mix to compensate for additional volume
from the UNG.
§ Applied Biosystems recommends that no more than 20% of the PCR be composed of the reverse
transcription reaction.
# Replicate volumes include 20% excess to compensate for volume loss from pipetting.
2. Cap the tube and invert it several times to mix the reaction components.
3. Centrifuge the tube briefly.
Load the plate

1. Transfer 20 µL of PCR reaction mix into each well of a 48-, 96-, or 384-well reaction
plate.
See Table 12 on page 42 for a list of compatible reaction plates and accessories.
2. Seal the plate with the appropriate cover.
3. Centrifuge the plate briefly.
4. Load the plate into the instrument.


Create the plate document/experiment and run the plate
1. Create a plate document/experiment for the run using the parameter values
shown in Table 3.
2. Run the plate.

Instructions on how to create and run a plate document/experiment are on
page 67 in “Related documentation”, which provides a list of resource documents
for your instrument.
Table 3 Plate document/experiment parameters for TaqMan® Gene Expression Assays
Thermal cycling conditions

Plate document/
Reaction
System Run experiment
plate Temp Time
parameters Stage
(°C) (mm:ss)
Applied Biosystems 7300/7500 Standard 96-well • Rxn. Volume: 20 µL Hold§ 50 2:00

Real-Time PCR System standard • Ramp Rate: Hold 95 10:00
Standard‡
Cycle 95 0:15
(40 Cycles) 60 1:00
Applied Biosystems 7500 Fast Standard 96-well • Rxn. Volume: 20 µL Hold§ 50 2:00
Real-Time PCR System Fast • Ramp Rate: Hold 95 10:00
Standard
Cycle 95 0:15
(40 Cycles) 60 1:00
Fast 96-well • Rxn. Volume: 20 µL Hold§ 50 2:00
Fast • Ramp Rate: Fast Hold 95 0:20
Cycle 95 0:03
(40 Cycles) 60 0:30
Applied Biosystems 7900HT Standard 96-well • Rxn. Volume: 20 µL Hold§ 50 2:00
Standard
384-well • Rxn. Volume: 20 µL Cycle 95 0:15
standard • Ramp Rate: (40 Cycles) 60 1:00
Standard
Fast • Ramp Rate: Fast Hold 95 0:20
384-well • Rxn. Volume: 20 µL Cycle 95 0:01
standard • Ramp Rate: Fast (40 Cycles) 60 0:20

Thermal cycling conditions

Plate document/
Reaction
System Run experiment
plate Temp Time
parameters Stage
(°C) (mm:ss)
Applied Biosystems StepOne™/ Standard 48-/96- • Rxn. Volume: 20 µL Hold§ 50 2:00

StepOnePlus™ Real-Time PCR well Fast • Ramp Speed: Hold 95 10:00
System Standard
Cycle 95 0:15
(40 Cycles) 60 1:00
Fast 48-/96- • Rxn. Volume: 20 µL Hold§ 50 2:00
well Fast • Ramp Speed: Fast Hold 95 0:20
Cycle 95 0:01
(40 Cycles) 60 0:20
Applied Biosystems ViiA™ 7 Standard 384-well • Rxn. Volume: 20 µL Hold§ 50 2:00
Standard
Cycle 95 0:15
(40 Cycles) 60 1:00
standard • Ramp Rate: Fast Hold 95 0:20
Cycle 95 0:01
(40 Cycles) 60 0:20
‡ The 7300 system has only one run mode (Standard 7300).
§ Required for optimal UNG activity; not needed when UNG is not in the reaction.

Analyze the results
Analyze the results

Analyzing the data from TaqMan Gene Expression Assays requires you to:
• View the amplification plots for the entire plate.
• Set the baseline and threshold values.
• Use the relative standard curve or the comparative CT method to analyze your
data.
Resources for data analysis

The details of data analysis depend on the real-time PCR instrument that you use; refer
to the appropriate user guide for instructions on how to analyze your data.
Table 4 Data analysis guides for Applied Biosystems real-time PCR systems
Real-time Part
Document
PCR system number
7900HT Fast Relative Quantitation Using Comparative CT Getting Started Guide 4364016
system
Performing Fast Gene Quantification: Quick Reference Card 4351892
Performing Fast Gene Quantitation with 384-Well Plates: User 4369584
Bulletin
7300/7500/ Relative Quantification: Getting Started Guide 4347824
7500 Fast
Relative Standard Curve and Comparative CT Experiments Getting 4387783
system
Started Guide
StepOne™/ Comparative CT/Relative Standard Curve and Comparative CT 4376785
StepOnePlus™ Experiments Getting Started Guide
system
All Applied Biosystems 7900HT Fast Real-Time PCR Systems and 4348358
7300/7500/7500 Fast Real-Time PCR Systems Chemistry Guide

Analyze the results
Tools for data analysis

Applied Biosystems recommends the following software for analyzing data generated
using TaqMan® Gene Expression Assays.
DataAssist™ DataAssist™ Software is a simple, yet powerful data analysis tool for sample
Software comparison when using the comparative CT (ΔΔCT) method for calculating relative
quantitation of gene expression. It contains a filtering procedure for outlier removal,
various normalization methods based on single or multiple genes, and provides
relative quantification analysis of gene expression through a combination of statistical
analysis and interactive visualization.
DataAssist™ Software is free and can be downloaded from:
www.appliedbiosystems.com/dataassist
RealTime RealTime StatMiner® Software from Integromics is a software analysis package for
StatMiner® qPCR experiments that is compatible with all Applied Biosystems instruments.
Software RealTime StatMiner® Software uses a step-by-step analysis workflow guide that
includes parametric, non-parametric, and paired tests for relative quantification of
gene expression, as well as 2-way ANOVA for two-factor differential expression
analysis.
For more information, visit:
www.integromics.com/StatMiner

Troubleshooting
Troubleshooting
Observation Possible cause Recommended action
Amplification curve shows The baseline was set Refer to your real-time PCR system user guide for
abnormal plot and/or low ΔRn improperly (some samples procedures on setting the baseline.
values. have CT values lower than the
Switch from manual to automatic baselining, or move
baseline stop value).
Linear view: the baseline stop value to a lower CT (2 cycles before
Amplification Plot the amplification curve for the sample crosses the
2.500
threshold).
2.000 Log view corrected:
Amplification Plot
1.000 E+1
Rn
1.500
1.000
1.000
1.000 E-1
5.000 E1
Rn
1.000 E-2
0 5 10 15 20 25 30 35 40
Cycle
1.000 E-3
Log view: 1.000 E-4
Amplification Plot
1.000 E+1
1.000 E-5
0 5 10 15 20 25 30 35 40
Cycle
1.000
1.000 E-1 An amplification signal is Dilute the sample to increase the CT value.
detected in the early cycles
Rn
1.000 E-2
(no baseline can be set

1.000 E-3
because the signal is detected

1.000 E-4
too early).
1.000 E-5
0 5 10 15 20 25 30 35 40
Cycle
Amplification curve shows a There is interaction between • Adjust the threshold manually.
rising baseline. the primer and probe. • Select another assay from the same gene, if
Linear view: available.
Amplification Plot
1.000
1.000
1.000
Rn
1.000
1.000
1.000
0 5 10 15 20 25 30 35 40
Cycle
Log view:
Amplification Plot
1.000 E+1
1.000
1.000 E-1
Rn
1.000 E-2
1.000 E-3
0 5 10 15 20 25 30 35 40
Cycle

Troubleshooting
Amplification curve shows (Custom TaqMan Gene Perform bioinformatics analysis. For more information,
weak amplification. Expression Assays only) refer to the:
Sequence provided for the • Bioinformatic Evaluation of a Sequence for Custom
assay design contains TaqMan® Gene Expression Assays Tutorial
mismatches with sample (PN 4371002).
sequences.
• Custom TaqMan® Assays Design and Ordering Guide
(PN 4367671).
Reagents and/or probe are • Check the expiration date of the reagents.
degraded reagents. • Verify that you follow the correct handling and
storage conditions.
• Avoid excessive freeze-thaw cycles. (Consider
diluting the 60✕ TaqMan® Gene Expression Assay
to a 20✕ working stock.)
Template is contaminated or • Improve the sample integrity (extraction methods).
degraded. See “Prepare the cDNA sample” on page 15.
• Check each template preparation by agarose gel
electrophoresis or bioanalyzer to determine the:
– Purity (only one product should be formed)
– Level of degradation
• Use RNase-free, sterile, filtered water.
Inhibitors are present in the Verify the presence of an inhibitor:
reaction.
1. Create a serial dilution of your sample.
2. Run the serial dilution with an assay for an
expressed gene (for example, an endogenous
control). If an inhibitor is present, low concentrations
yield higher-than-expected CT values. (High
concentration means more inhibition because the
sample is not diluted.)
3. Rerun the assay with purified template.
4. Improve the sample integrity (extraction methods).
See “Prepare the cDNA sample” on page 15.
The reverse transcription (RT) • Check the RNA sample for degradation.
conversion to cDNA is poor. • Input RNA could be too concentrated or too dilute.
Verify the concentration by optical density (OD),
make new serial dilutions of template RNA from the
original stock, then repeat the RT-PCR.
• Ensure that the RT-PCR setup is performed under
the appropriate conditions to avoid premature cDNA
synthesis.
• Check the RT reagents for contamination and/or
degradation.
Primer-dimer formation and (Fast chemistry only) For optimal results, run the
residual polymerase activity reaction plate as soon as possible after completing the
occurs. reaction setup. If you cannot run a reaction plate within
2 hours after completing the reaction setup, refrigerate
or freeze the reaction plate until you can run it.

Troubleshooting
Amplification curve shows low Inaccurate pipetting: Little or Follow accurate pipetting practices.
ROX™ dye (passive reference no TaqMan® Universal PCR
dye). Master Mix is present.
Amplification curve shows no One or more of the reaction Verify that the cDNA, TaqMan Gene Expression Assays,
amplification of the sample components was not added. and TaqMan® Gene Expression Master Mix were added
(CT = 40) across all assays or to the reaction plate. (If the master mix is missing, the
in an unusually large number passive reference fails.)
of assays.
Incorrect dye components Check the dye components settings and reanalyze the
were selected. data.
The annealing temperature on Verify that the thermal cycler is set to the correct
the thermal cycler was too annealing and extension temperatures. Ensure that the
high for the primers and/or thermal cycler is calibrated and maintained regularly.
probe.
Inappropriate reaction Troubleshoot the RT-PCR optimization.
conditions were used.
The template is degraded. • Determine the quality of the template.
• Rerun the assay with fresh template.
• Use RNase-free reagents.
• Use an RNase inhibitor.
Inhibitors are present in the Verify the presence of an inhibitor:
reaction.
1. Create a serial dilution of your sample.
2. Run the serial dilution with an expressing assay (for
example, an endogenous control). If an inhibitor is
present, low concentrations yield higher-than-
expected CT values. (High concentration means
more inhibition because the sample is not diluted.)
3. Rerun the assay with purified template.
The baseline and/or threshold Refer to your real-time PCR system user guide for
was improperly set. procedures on setting the baseline and threshold:
• Switch from automatic to manual baselining, or
from manual to automatic.
• Lower the threshold value to within the appropriate
range.
cDNA conversion failed. • Check the RNA integrity and concentration.
• Check for RNase activity.
• Follow Applied Biosystems recommended thermal
profile.
• Repeat the RT step using new reagents.

Troubleshooting
Amplification curve shows no (Custom TaqMan Gene • Verify that the sequence that you submitted is
amplification of the sample Expression Assays only) Assay correct.
(CT = 40) across all assays or design or synthesis failure: • Check for an alternative transcript or a splice
in an unusually large number The wrong sequence was variant.
of assays. submitted to Applied
Biosystems.
(Custom TaqMan Gene Verify that the location targeted by the assay is not
Expression Assays only) Assay within the 5′ untranslated region (UTR), which can be
is designed in a variable highly variable between transcripts.
region of the gene transcript.
If the assay is designed within the 5′ UTR, select a
different assay that is within the coding region of the
transcript. Otherwise, select an assay for an alternative
transcript or splice variant.
Amplification curve shows The baseline was set Refer to your real-time PCR system user guide for
samples targeted by the same improperly. procedures on setting the baseline:
assay that have differently • Switch from automatic to manual baselining, or
shaped curves. from manual to automatic.
• Increase the upper or lower value of the baseline
range.
Sample quality is poor. 1. Perform a quality check on the sample.
2. If necessary, reextract the sample.
Different concentrations Follow accurate pipetting practices.
caused by imprecise pipetting.
Reagents or equipment is Be sure that your workspace and equipment are
contaminated. properly cleaned.

Troubleshooting
Amplification curve shows no The gene is not expressed in • Verify the known expression of the gene in the
amplification of the sample the tested sample. sample type.
(CT = 40) in the target assay. • Verify by:
– Rerunning the sample using the same assay.
– Rerunning the assay using more sample. Avoid
preparing the PCR reaction mix with more than
20% from the reverse transcription reaction.
– (TaqMan Gene Expression Assays only) Running the
sample using an alternative assay, if available,
that detects a different transcript or more than
one transcript from the same gene.
Note: If the recommended actions do not
resolve the problem, the result may be correct.
The sample may not have Verify by:
enough copies of the target • Rerunning the sample using the same assay.
RNA.
• Rerunning the assay using more sample. Avoid
preparing the PCR reaction mix with more than 20%
from the reverse transcription reaction.
Note: If the recommended actions do not
resolve the problem, the result may be correct.
One or more of the reaction Check your pipetting equipment and/or technique.
components was not added.
Incorrect dye components Check the settings of the dye components before data
were selected. analysis.
Decrease in ROX™ dye Precipitation in the TaqMan® • When using the TaqMan® PCR Core Reagents Kit,
fluorescence (passive buffers occurs. be sure to mix the tubes well.
reference dye). • Use TaqMan® Gene Expression Master Mix (2✕). Be
sure to mix thoroughly to produce a homogenous
solution.
Reagents are degraded. Verify that kits and reagents have been stored
according to the instructions on the packaging and that
they have not expired.
Simultaneous increase in The sample evaporated. Check the seal of the optical adhesive cover for leaks.
fluorescence from both the:
• Passive reference (ROX™)
dye
• Reporter dye(s)
Multicomponent signal for Pure dye components spectra Rerun the pure dye spectra.
ROX™ dye is not linear. are incorrect.
Incorrect dye components Select the correct dyes for the data analysis.
were selected.
Rn on Rn-vs.-Cycle plot is very ROX™ dye was not selected as Select the ROX™ dye as the passive reference, then
high. the passive reference when reanalyze the data.
the plate document/
experiment was set up.

Troubleshooting
No template control (NTC) Contaminated reagents • Rerun the assay using new reagents.
shows amplification. (contaminated with gDNA, • Be sure your workspace and equipment are cleaned
amplicon, or plasmid clones). properly.
• Use UNG.
• Run no-reverse-transcription controls to rule out
genomic DNA contamination.
• Treat the sample with DNase.
• (gDNA contamination only; TaqMan Gene Expression
Assays only) Design an assay that spans an exon-
exon boundary.
(TaqMan Gene Expression Use AmpliTaq Gold® LD DNA Polymerase.
Assays only) Bacterial
sequences used as template.
The endogenous control CTs Endogenous control is not Refer to the Application Note: Using TaqMan®
vary, or do not normalize the consistently expressed across Endogenous Control Assays to Select an Endogenous
sample well. the samples. Control for Experimental Studies (127AP08-01) for
information on selecting an endogenous control.
Sample concentrations vary If desired, quantitate and normalize samples before
widely. running them.
Pipetting was inaccurate. • Check the calibration of the pipettes.
• Pipet more than 5 µL of sample.
High standard deviation of Inefficient mixing of reagents. • Increase the length of time that you mix the
replicates (inconsistent data, reagents.
CT varies). • Validate your mixing process by running a replicate
plate.
Pipetting was inaccurate. • Check the calibration of the pipettes.
• Pipette more than 5 µL of sample.
Threshold was set improperly. Set the threshold above the noise and where the
replicates are tightest. Refer to your real-time PCR
system user documentation for procedures on setting
the threshold.
Low concentration of target. Rerun the assay using more template.
Template absorption occurred Add a carrier (for example, yeast tRNA).
(adhering to the tube).

Troubleshooting
CT value is lower than gDNA contamination • Verify contamination by running an RT-minus

expected. occurred. reaction (without the reverse transcriptase).
• (TaqMan Gene Expression Assays only) Perform
bioinformatics analysis: design a custom assay to
span an exon-exon junction. For more information,
refer to the:
– Bioinformatic Evaluation of a Sequence for Custom
TaqMan® Gene Expression Assays Tutorial
(PN 4371002).
– Custom TaqMan® Assays: Design and Ordering
Guide (PN 4367671).
More sample added than • Reduce the amount of sample.
expected. • Quantitate and adjust the concentration of the
sample.
Template or amplicon is Follow established PCR good laboratory practices.
contaminated.
Amplification occurs in the gDNA contamination • Improve sample extraction methods to eliminate
no-RT controls. occurred. gDNA. See “Isolate total RNA” on page 15.
• (TaqMan Gene Expression Assays only) Perform
bioinformatics analysis: design a custom assay to
span an exon-exon junction. For more information,
refer to the:
– Bioinformatic Evaluation of a Sequence for Custom
TaqMan® Gene Expression Assays Tutorial
(PN 4371002).
– Custom TaqMan® Assays: Design and Ordering
Guide (PN 4367671).
Template or amplicon is Follow established PCR good laboratory practices.
contaminated.
Shifting Rn value during the Fluorescence did not stabilize • Reset the lower value of the baseline range.
early cycles of the PCR to the buffer conditions of the • Use automatic baselining.
(cycles 0 to 5). reaction mix.
Note: This condition
does not affect PCR or
the final results.
Small ΔRn. PCR efficiency is poor. Recheck the concentration of the reagents.
Quantity of starting target is Increase the quantity of the starting target.
low (low copy number of
target).

Troubleshooting
Noisy signal above the The sample evaporated. Check the seal of the optical adhesive cover for leaks.
threshold.
The well is empty because of • Check the calibration of the pipettes.
inaccurate pipetting. • Pipet more than 5 µL of sample.
The well is labeled with a • Be sure that your plate document/experiment is set
detector in the plate up correctly.
document/experiment, but the • Exclude the well and reanalyze the data.
well is empty.

Troubleshooting

APPENDIX A
How to Order TaqMan® Gene Expression

Assays
To order TaqMan® Gene Expression Assays, follow these steps:

■ Step 1: Search for an assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
■ Step 2: Order the assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
■ Step 3: Order a candidate endogenous control assay . . . . . . . . . . . . . . . . . . . . . . . 36
■ Step 4: Select a chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
■ Step 5: Order materials and equipment not included. . . . . . . . . . . . . . . . . . . . . . . 41

Appendix A How to Order TaqMan® Gene Expression Assays
Step 1: Search for an assay

Search for TaqMan® Gene Expression Assays or Non-coding RNA Assays
To perform a full assay search:
1. Go to www.appliedbiosystems.com.
2. Place the cursor over Products, then select TaqMan Gene Expression Assays
under Assay Searches.
3. At the TaqMan Gene Expression Assays search page, select TaqMan Gene
Expression Assays, TaqMan Non-coding RNA Assays, or All Assays from the
pull-down menu.
4. Search for assays that match your target of interest.

To learn more about how to order using the full assay search, refer to the Online
Ordering Guide for TaqMan® Gene Expression Assays (SN 127MI07-05). To select the
ideal assay for your research, refer to the Online Selection Guide for TaqMan® Gene
Expression Assays (SN 127GU08-01).
Search for TaqMan® Gene Expression Assays only

Table 5 Search tools and methods for TaqMan® Gene Expression Assays
Method Description
Perform a quick 1. Go to www.appliedbiosystems.com.

search 2. Under “I Want to Buy,” select TaqMan Gene Expression Assays,
Plates & Arrays.
3. In the Gene Expression Assays, Plates & Arrays page, select
TaqMan® Gene Expression Assays.
4. Use the orange Start Here box to search for assays that match your
gene of interest.
GeneAssist™ Go to www4.appliedbiosystems.com/tools/pathway, then follow the
Pathway Atlas instructions to search the GeneAssist™ Pathway Atlas.
This tool provides access to more than 350 interactive cell signaling
maps, and enables you to view gene and disease information while
you order Silencer® siRNAs and corresponding TaqMan® Gene
Expression Assays.
UMapIt™ Mapping Go to www4.appliedbiosystems.com/tools/umapit, then follow the
Tool instructions to find and order TaqMan® Gene Expression Assays for
validating microarray hits or for performing follow-up experiments.
This tool provides an easy way to find TaqMan® assays from
microarray probe IDs.

About TaqMan® Gene Expression Assay IDs

Note: The assay ID prefix and suffix codes for TaqMan Gene Expression Assays
and TaqMan Non-coding RNA Assays are identical.
The assay ID prefix indicates the species to which the assay is designed.
Table 6 Assay ID prefix codes
Prefix Species
At Arabodopsis thaliana
Bt Bos taurus (Cow)
Ce Caenorhabditis elegans
Cf Canis familiaris (Dog)
Cp Cavia. porcellus (Guinea Pig)
Dm Drosophila melanogaster
Dr Danio rerio (Zebrafish)
Ec Equus caballus (Horse)
Gg Gallus gallus (Chicken)
Gm Glycine max (Soybean)
Hs Homo sapiens
Mm Mus musculus
Oc Oryctolagus cuniculus (Rabbit)
Os Oryza sativa (Rice)
Rh Macaca mulatta (Rhesus)
Rn Rattus norvegicus
Sp Schizosaccharomyces pombe (Fission Yeast)
Ss Sus scroga (Pig)
Xt Xenopus tropicalis (Frog)

The assay ID suffix indicates the assay placement.
Table 7 Assay ID suffix codes
Suffix Definition
_m An assay whose probe spans an exon junction.

_s An assay whose primers and probes are designed within a single exon. Such
assays, by definition, detect genomic DNA.
_g An assay that may detect genomic DNA. The assay primers and probe may also be
within a single exon.
_mH An assay that is designed to a transcript belonging to a gene family that has high
sequence homology. The assays are designed to yield a 10- to 15-CT difference
_sH
between the target gene and the gene with the closest sequence homology. This
_gH means that an assay detects the target transcript with 1000- to 30,000-fold greater
discrimination (sensitivity) than the closest homologous transcript, if both
transcripts are at the same copy number in a sample.
_u An assay whose amplicon spans an exon junction, and whose probe binds
completely in one of the spanned exons.
_ft An assay designed to detect fusion transcripts that result from chromosomal
translocation. One primer and the probe are on one side of the fusion transcript
breakpoint, and the second primer is on the other side of the fusion transcript
breakpoint. The assay does not detect gDNA.
_at An assay that is designed to detect a synthetic RNA transcript with a unique
sequence that lacks homology to current annotated biological sequences.
Note: An assay ID beginning with “Hs999999…” and ending in “_m1”

identifies a TaqMan® Gene Expression Assay that amplifies a region spanning
an exon junction, although the associated probe does not span the junction. For
example, the exon boundary information for assay Hs99999903_m1 is 1-1,
indicating that the probe targets a region within exon 1, not the exon junction
itself. Although the probe binds within a single exon, the amplicon spans exons
1-2 (the forward primer and probe are in exon 1, but the reverse primer is in
exon 2).

Step 2: Order the assay
Step 2: Order the assay

Order TaqMan® Gene Expression Assays and TaqMan® Non-coding RNA Assays
TaqMan Gene Expression Assays are available in a variety of formulations. For a
comprehensive list of available assays and part numbers, please refer to Table 1 on
page 10. All of the assay search methods also provide methods for ordering assays.
If you cannot find a predesigned assay to suit your needs, you can order Custom
TaqMan® Gene Expression Assays as described on this page.
Order Custom TaqMan® Expression Assays

Use the Custom TaqMan Assay Design Tool (www.appliedbiosystems.com/cadt) to
enter and submit sequences for new assay design. The tool also supports submission
files created or validated using File Builder software. For details, refer to the Custom
TaqMan® Assays: Design and Ordering Guide (PN 4367671).
Reorder a Custom TaqMan® Gene Expression Assay

Reorder Custom TaqMan Gene Expression Assays through the Custom TaqMan Assay
Design Tool.
1. At www.appliedbiosystems.com/cadt, select Reorder Existing Custom Assays.
2. In the Search tab, search for existing custom assays by:

• Assay ID: the ID assigned to a custom assay ordered after Jan 2009. Format:
seven alphanumeric characters.
• Legacy Assay ID: the ID assigned to a custom assay ordered before February
2009. Format: sequence name-target name.
• Assay Name: the sequence name of any assay designed using the Custom
TaqMan Assay Design Tool.
• Date Range.
• Sales Order #.
The Custom TaqMan® Assays: Design and Ordering Guide (PN 4367671) has details on
reordering legacy assays and those designed in the Custom TaqMan® Assay Design
Tool, including reordering by e-mail and regular or express mail.

Step 3: Order a candidate endogenous control assay

Search for and order the endogenous control assay using one of the methods described
in Step 1 on page 32.
Assays for normalization

A valid normalization or endogenous control is needed to correct for differences in
RNA sampling and sample variation. The ideal control is expressed consistently under
experimental conditions and is sufficiently abundant across all tissues and cell types
studied.
Note: Applied Biosystems recommends that you experimentally validate all
candidate genes to be used as endogenous controls.
Use Table 8 to order… Use Table 9 on page 38 to order…
Non-primer limited, FAM™ dye-labeled • Non-primer limited, FAM™ dye-labeled MGB

MGB probe (250 20-µL reactions; probe (other sizes)
PN 4331182) • Primer limited, VIC® dye-labeled MGB probe
• Primer limited, VIC® dye-labeled, TAMRA™
dye-quenched probe
Table 8 Candidate endogenous control assays (non-primer limited; PN 4331182)
Gene
Gene name Human assay ID Mouse assay ID Rat assay ID
symbol
18S Eukaryotic 18S rRNA Hs99999901_s1 Hs99999901_s1 Hs99999901_s1

ABL1 C-abl oncogene 1, receptor tyrosine kinase Hs00245445_m1 Mm00802029_m1 Rn01436238_g1
ACTB Actin, Beta, cytoplasmic Hs99999903_m1 Mm00607939_s1 Rn00667896_m1
B2M Beta-2-microglobulin Hs99999907_m1 Mm00437762_m1 Rn00560865_m1
CASC3 Cancer susceptibility candidate 3 Hs00201226_m1 Mm00454629_m1 Rn00595941_m1
CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Hs00355782_m1 Mm00432448_m1 Rn00589996_m1
Cip1)
CDKN1B Cyclin-dependent kinase inhibitor 1B (p27, Hs00153277_m1 Mm00438168_m1 Rn00582195_m1
Kip1)
EIF2B1 Eukaryotic translation initiation factor 2B, Hs00426752_m1 Mm00460997_m1 Rn00596951_m1
subunit 1 alpha, 26kDa
ELF1 E74-like factor 1 (ETS-domain transcription Hs00152844_m1 Mm00468217_m1 Rn00585356_m1
factor)
GADD45A Growth arrest and DNA-damage-inducible, Hs00169255_m1 Mm00432802_m Rn00577049_m1
alpha
GAPDH Glyceradehyde-3-phosphate dehydrogenase Hs99999905_m1 Mm99999915_g1 Rn99999916_s1
GUSB Beta glucuronidase Hs99999908_m1 Mm00446953_m1 Rn00566655_m1
HMBS Hydromethylbilane synthase Hs00609297_m1 Mm00660262_g1 Rn00565886_m1

Gene
Gene name Human assay ID Mouse assay ID Rat assay ID
symbol
HPRT1 Hypoxanthine guanine phosphoribosyl Hs99999909_m1 Mm00446968_m1 Rn01527840_m1

transferase 1
IP08 Importin 8 Hs00183533_m1 Mm01255158_m1 Rn00821065_g1
MRPL19 Mitochondrial ribosomal protein L19 Hs00608519_m1 Mm00452754_m1 Rn01425270_m1
MT-ATP6 Mitochondrially encoded ATP synthase 6 Hs02596862_g1 Mm03649417_g1 Rn03296710_s1
PES1 Pescadillo homolog 1, containing BRCT Hs00362795_g1 Mm00727566_s1 Rn01443731_g1
domain (zebrafish)
PGK1 Phosphoglycerate kinase 1 Hs99999906_m1 Mm00435617_m1 Rn00821429_g1
POLR2A Polymerase (RNA) II (DNA directed) Hs00172187_m1 Mm00839493_m1 Rn00574762_m1
polypeptide A, 220 kDa
POP4 Processing of precursor 4, ribonuclease P/ Hs00198357_m1 Mm00481282_m1 Rn02347225_m1
MRP subunit (S. cerevisiae)
PPIA Peptidylprolyl isomerase A Hs99999904_m1 Mm02342430_g1 Rn00690933_m1
PSMC4 Proteasome (prosome, macropain) 26S Hs00197826_m1 Mm00457191_m1 Rn00821605_g1
subunit, ATPase, 4
PUM1 Pumilio homolog 1 (Drosophila) Hs00206469_m1 Mm00472886_m Rn00982780_m1
RPL30 Ribosomal protein L30 Hs00265497_m1 Mm01611464_g1 Rn01504620_g1
RPL37A Ribosomal protein L37a Hs01102345_m1 Mm01546394_s1 Rn02114291_s1
RPLP0 Ribosomal protein, large, P0 Hs99999902_m1 Mm00782638_s1 Rn01479927_g1
RPS17 Ribosomal protein S17 Hs00734303_g1 Mm01314921_g1 Rn00820807_g1
TBP TATA box binding protein Hs99999910_m1 Mm00446973_m1 Rn01455648_m1
TFRC Transferrin receptor Hs99999911_m1 Mm00441941_m1 Rn01474695_m1
UBC Ubiquitin C Hs00824723_m1 Mm01201237_m1 Rn01789812_g1
YWHAZ Tyrosine 3-monooxygenase, or tryptophan 5- Hs00237047_m1 Mm01158417_g1 Rn00755072_m1
monooxygenase activation protein, zeta
polypeptide

Table 9 TaqMan® Endogenous Controls (additional formulations)
Corresponding
No. of
Gene Reporter Primer Part TaqMan® Assay
Species Gene name Quencher 20-µL
symbol dye limited number ID in Table 8
rxns.
(PN 4331182)
Eukaryotic 18S 18S ribosomal FAM™ MGB No 4333760T 125 Hs99999901_s1

RNA
FAM™ MGB No 4333760F 500 Hs99999901_s1
FAM™ MGB No 4352930E 2500 Hs99999901_s1
VIC® MGB Yes 4319413E 2500 N/A
VIC® TAMRA™ Yes 4310893E 2500 N/A
Human ACTB Beta actin FAM™ MGB No 4333762T 125 Hs99999903_m1
FAM™ MGB No 4333762F 500 Hs99999903_m1
FAM™ MGB No 4352935E 2500 Hs99999903_m1
B2M Beta-2- FAM™ MGB No 4333766T 125 Hs99999907_m1
microglobulin
FAM™ MGB No 4333766F 500 Hs99999907_m1
GAPDH Glyceraldehyde FAM™ MGB No 4333764T 125 Hs99999905_m1
-3-phosphate
FAM™ MGB No 4333764F 500 Hs99999905_m1
dehydrogenase
FAM™ MGB No 4352934E 2500 Hs99999905_m1
GUSB Beta FAM™ MGB No 4333767T 125 Hs99999908_m1
glucuronidase
FAM™ MGB No 4333767F 500 Hs99999908_m1
HPRT1 Hypoxanthine- FAM™ MGB No 4333768T 125 Hs99999909_m1
phosphoribosyl
FAM™ MGB No 4333768F 500 Hs99999909_m1
transferase 1
PGK1 Phosphoglycer FAM™ MGB No 4333765T 125 Hs99999906_m1
ate kinase 1
FAM™ MGB No 4333765F 500 Hs99999906_m1

Corresponding
No. of
Gene Reporter Primer Part TaqMan® Assay
Species Gene name Quencher 20-µL
symbol dye limited number ID in Table 8
rxns.
(PN 4331182)
Human PPIA Cyclophilin A FAM™ MGB No 4333763T 125 Hs99999904_m1

FAM™ MGB No 4333763F 500 Hs99999904_m1
RPLP0 Ribosomal FAM™ MGB No 4333761T 125 Hs99999902_m1
protein, large,
FAM™ MGB No 4333761F 500 Hs99999902_m1
P0
TBP TATA-box FAM™ MGB No 4333769T 125 Hs99999910_m1
binding protein
FAM™ MGB No 4333769F 500 Hs99999910_m1
TFRC Transferrin FAM™ MGB No 4333770T 125 Hs99999911_m1
receptor (p90,
FAM™ MGB No 4333770F 500 Hs99999911_m1
CD71)
Mouse ACTB Beta actin FAM™ MGB No 4352933E 2500 Mm00607939_s1
GAPDH Glyceraldehyde FAM™ MGB No 4352932E 2500 Mm99999915_g1
-3-phosphate
dehydrogenase
Rat ACTB Beta actin FAM™ MGB No 4352931E 2500 Rn00667869_m1
GAPDH Glyceraldehyde FAM™ MGB No 4352936E 2500 Rn99999916_s1
-3-phosphate
dehydrogenase

Step 4: Select a chemistry
Step 4: Select a chemistry

Select standard or Fast chemistry
StepOne™, StepOnePlus™, 7500 Fast, 7900HT, and ViiA™ 7 Fast Real-Time PCR
Systems contain Fast thermal cycling blocks that can perform Fast quantitative PCR.
Applied Biosystems Fast PCR systems use high-speed thermal cycling blocks,
TaqMan® Fast Advanced Master Mix, and optical Fast thermal cycling plates and tubes
to reduce quantitative PCR run times to less than 40 minutes. For more information on
Fast chemistries available from Applied Biosystems, refer to the Data Sheet: Comparing
Fast and Standard Data on Applied Biosystems 7500 and 7500 Fast Real-Time PCR Systems
(SN 117MI08-01).
Table 10 Chemistry and plates for a standard or Fast run
Component Standard chemistry per run Fast chemistry per run
cDNA quantity 1–100 ng 1–100 ng

TaqMan® • TaqMan® Gene Expression TaqMan® Fast Advanced Master
Master Mix Master Mix Mix
• TaqMan® Universal Master Mix
II, with or no UNG
• TaqMan® Universal PCR Master
Mix (2✕), with or without
AmpErase® UNG
96-well plate MicroAmp® Optical 96-Well MicroAmp® Fast Optical 96-Well
Reaction Plate with Barcode Reaction Plate with Barcode
384-well plate MicroAmp® Optical 384-Well Reaction Plate with Barcode
48-well plate MicroAmp® Fast Optical 48-Well Reaction Plate
Select 1- or 2-step RT-PCR

Applied Biosystems offers several chemistries that you can use to perform RT-PCR in
one or two steps. See Table 11 on page 41 for a list of kits.

Step 5: Order materials and equipment not included

See “Materials for reverse transcription and PCR” on page 12 for a list of required
materials.
Related reagents
Table 11 Reagents for reverse transcription and PCR
Reagent Description and part number
TaqMan® Gene Expression Master Mix • One 1-mL tube (PN 4370048)
(2✕) • One 5-mL bottle (PN 4369016)
• One 6-mL bottle (PN 4393469)
• Two 5-mL bottles (PN 4369514)
• Five 5-mL bottles (PN 4369510)
• Ten 5-mL bottles (PN 4369542)
TaqMan® Universal Master Mix II, no UNG • One 1-mL tube (PN 4440043)
TaqMan® Universal Master Mix II, with • One 1-mL tube (PN 4440042)
UNG • One 5-mL bottle (PN 4440038)
TaqMan® Universal PCR Master Mix (2✕) • One 5-mL bottle (PN 4304437)
TaqMan® Universal Master Mix (2✕) No • One 5-mL bottles (PN 4324018)
AmpErase® UNG • Two 5-mL bottles (PN 4364341)

Reagent Description and part number
TaqMan® Fast Advanced Master Mix • Mini-Pack 1 ×1 mL 100 (PN 4444556)

• 1-Pack 1× 5 mL 500 (PN 4444557)
• 2-Pack 2 × 5 mL 1,000 (PN 4444963)
• 5-Pack 5 × 5 mL 2,500 (PN 4444964)
• 10-Pack 10 × 5 mL 5,000 (PN 4444965)
• Bulk-Pack 1 × 50 mL 5,000 (PN 4444558)
TaqMan®Fast Universal Master Mix (2✕) • 250 × 20-µL reactions (PN 4352042)
No AmpErase® UNG • 500 × 20-µL reactions (PN 4366072)
• 1250 × 20-µL reactions (PN 4366073)
• 2500 × 20-µL reactions (PN 4364103)
• 5000 × 20-µL reactions (PN 4367846)
High Capacity RNA-to-cDNA Kit 50 reactions (PN 4387406)
High Capacity cDNA Reverse • 200 reactions (PN 4368814)
Transcription Kit • 200 reactions with RNase Inhibitor
(PN 4374966)
• 1000 reactions (PN 4368813)
• 1000 reactions with RNase Inhibitor
(PN 4374967)
TaqMan® RNA-to-CT™ 1-Step Kit • 40 × 50-µL reactions (PN 4392653)
• 200 × 50-µL reactions (PN 4392938)
• 2000 × 50-µL reactions (PN 4392656)
Nuclease-free water (no diethyl 500 mL (PN AM9930)
pyrocarbonate [DEPC])
Reaction plates and accessories

Table 12 Reaction plates and accessories for Applied Biosystems thermal cyclers and real-
time PCR systems
Instrument Reaction plates and accessories
7300 system • MicroAmp® Optical 96-Well Reaction Plate with Barcode:

7500 system – 500 plates (PN 4326659)
– 20 plates (PN 4306737)
• MicroAmp® Optical Adhesive Film (PN 4311971)
• MicroAmp® Optical 8-Tube Strips, 0.2-mL, 1000 tubes in strips of
eight (PN 4316567)
• MicroAmp® Optical 8-Cap Strips, 300 strips (PN 4323032)
7500 Fast system • MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode:
– 200 plates (PN 4366932)
– 20 plates (PN 4346906)

7900HT Fast system, • MicroAmp® Optical 96-Well Reaction Plate with Barcode:
standard 96-well – 500 plates (PN 4326659)
block
– 20 plates (PN 4306737)
• MicroAmp® Optical Film Compression Pad (PN 4312639) for use
with one plate
7900HT Fast system, • MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode:
Fast 96-well block – 200 plates (PN 4366932)
– 20 plates (PN 4346906)
• MicroAmp® Optical Film Compression Pad (PN 4312639) for use
with one plate
7900HT Fast system, • MicroAmp® Optical 384-Well Reaction Plate with Barcode:
384-well block – 1000 plates (PN 4343814)
– 500 plates (PN 4326270)
– 50 plates (PN 4309849)
• MicroAmp® Optical 384-Well Reaction Plate, 1000 plates
(PN 4343370)
Applied Biosystems • MicroAmp® Optical 384-Well Reaction Plate with Barcode:
ViiA™ 7 system – 1000 plates (PN 4343814)
– 500 plates (PN 4326270)
– 50 plates (PN 4309849)
• MicroAmp® Optical 384-Well Reaction Plate, 1000 plates
(PN 4343370)
StepOne™ system • MicroAmp® Fast Optical 48-Well Reaction Plate, 20 plates
(PN 4375816)
• MicroAmp® 48-Well Optical Adhesive Film (PN 4375323)
StepOnePlus™ • MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode:
system – 200 plates (PN 4366932)
– 20 plates (PN 4346906)

9700 instrument • MicroAmp® Optical 96-Well Reaction Plate with Barcode:

– 500 plates (PN 4326659)
– 20 plates (PN 4306737)
• MicroAmp® Optical 384-Well Clear Optical Reaction Plate with
Barcode:
– 1000 plates (PN 4343814)
– 500 plates (PN 4326270)
– 50 plates (PN 4309849)
• MicroAmp® Clear Adhesive Films, 100 films (PN 4306311)
• MicroAmp® Optical 8-Tube Strips, 0.2-mL, 1000 tubes in strips of
eight (PN 4316567)
• MicroAmp® Optical 8-Cap Strips, 300 strips (PN 4323032)
Veriti® 96-well • MicroAmp® Optical 96-Well Reaction Plate:
thermal cycler – 500 plates (PN 4316813)
– 10 plates (PN N8010560)
• MicroAmp® Clear Adhesive Films, 100 films (PN 4306311)
Veriti® 384-well • MicroAmp® Optical 384-Well Reaction Plate with Barcode:
thermal cycler – 1000 plates (PN 4343814)
– 500 plates (PN 4326270)
– 50 plates (PN 4309849)
Related gene expression assays and arrays products

Table 13 Related gene expression assays and arrays products
Assay or array For more information...
TaqMan® TaqMan® MicroRNA Assays miRNA.appliedbiosystems.com

assays
Custom TaqMan® Small RNA Assays www.appliedbiosystems.com
TaqMan® Pri-miRNA Assays mirna.appliedbiosystems.com

Custom TaqMan® Probes and www.appliedbiosystems.com
Primers‡

Assay or array For more information...
TaqMan® TaqMan® Array Cards: taqmanarray.appliedbiosystems.com

arrays • TaqMan® Custom Arrays
• TaqMan® Gene Signature Array
• TaqMan® Gene Sets
TaqMan® Array Plates§ www.allgenes.com
• TaqMan®MicroRNA Arrays miRNA.appliedbiosystems.com
– Human
– Rodent
• Megaplex™ Primer Pools
– Megaplex™ RT Primers
– Megaplex™ PreAmp Primers
‡ Probes and primers that are synthesized by Applied Biosystems to your exact sequence and choice of quencher
and reporter dyes.
§ TaqMan® Gene Expression Assays dried in MicroAmp® Optical 96-Well Reaction Plates.


APPENDIX B
Good PCR Practices
Prevent contamination and nonspecific amplification

PCR assays require special laboratory practices to avoid false positive amplifications.
The high throughput and repetition of these assays can lead to amplification of one
DNA molecule.
AmpErase® UNG
AmpErase® uracil-N-glycosylase (UNG) prevents reamplification of carryover-PCR
products in an assay if all previous PCR for that assay is performed using a dUTP-
containing master mix. UNG acts on single- and double-stranded dU-containing DNA
by hydrolyzing uracil-glycosidic bonds at dU-containing DNA sites. The enzyme
causes the release of uracil, thereby creating an alkali-sensitive apyrimidic site in the
DNA. The enzyme has no activity on RNA or dT-containing DNA (Longo et al., 1990).
PCR good laboratory practices

When preparing samples for PCR amplification:
• Wear clean gloves and a clean lab coat (not previously worn while handling
amplified PCR products or used during sample preparation).
• Change gloves whenever you suspect that they are contaminated.
• Maintain separate areas and dedicated equipment and supplies for:
– Sample preparation
– PCR setup
– PCR amplification
– Analysis of PCR products
• Never bring amplified PCR products into the PCR setup area.
• Open and close all sample tubes carefully. Try not to splash or spray PCR
samples.
• Keep reactions and components capped as much as possible.
• Use a positive-displacement pipette or aerosol-resistant pipette tips.
• Clean lab benches and equipment periodically with 10% bleach solution.

Appendix B Good PCR Practices
Prevent contamination and nonspecific amplification

APPENDIX C
Duplex Reactions
Duplex reactions using TaqMan® Gene Expression Assays

Duplex real-time PCR is the simultaneous amplification and measurement of two
target sequences in one reaction. TaqMan® Gene Expression Assays can be used in
duplex real-time PCR when using a FAM™ dye-labeled assay in combination with a
primer-limited, VIC® dye-labeled assay. However, Applied Biosystems strongly
recommends that you validate that your duplex assay combinations provide similar
results to your singleplex reactions. When setting a duplex reaction, it is important to:
• Consider the relative expression levels of each target.
• Perform serial dilutions of your sample in both singleplex and duplex reactions,
and compare the results for relative expression.
• Select the higher-expressing target as the primer-limited, VIC dye-labeled assay.
• Use TaqMan Gene Expression Master Mix, which has been optimized for
duplexing reactions.
For more details on how to validate your duplex assay reactions and interpret the
results, refer to the Applied Biosystems Application Note Factors Influencing Multiplex
Reactions (Publication 136AP04-01 O-081742), available at
www.appliedbiosystems.com.
Setting up a Duplex Reaction

A duplex reaction is run the same as a singleplex reaction, with the addition of the
primer-limited assay:
For each sample (to be run in quadruplicate) 1 reaction (µL) 4 reactions#
TaqMan Gene Expression Assay (20✕ FAM dye-labeled) 1 5

TaqMan Gene Expression Assay ( 20✕ VIC dye-labeled, primer-limited) 1 5
TaqMan Gene Expression Master Mix (2✕)‡ 10 50
cDNA Template (1-100ng)§ 4 20
RNAse-free water 4 20
‡ TaqMan Gene Expression Master Mix has been optimized for duplex reactions. Applied Biosystems does not recommend Universal
Master Mix or Fast Master Mix for duplex reactions.
§ Applied Biosystems recommends that the qPCR reaction not be composed of more than 20% of the reverse transcription reaction.
# Replicate reactions include 20% excess volume to account for losses during pipetting.
Instructions on how to create and run a plate document/experiment are on page 67 in

“Related documentation”, which provides a list of resource documents for your
instrument.

Appendix C Duplex Reactions
Duplex reactions using TaqMan® Gene Expression Assays

APPENDIX D
Background Information
About TaqMan® chemistry

About the probes
TaqMan® MGB probes contain:
• A reporter dye (for example, FAM™ dye) linked to the 5′ end of the probe.
• A minor groove binder (MGB) at the 3´ end of the probe.
MGBs increase the melting temperature (Tm) without increasing probe length
(Afonina et al., 1997; Kutyavin et al., 1997); they also allow for the design of
shorter probes.
• A nonfluorescent quencher (NFQ) at the 3′ end of the probe.
Because the quencher does not fluoresce, Applied Biosystems real-time PCR
systems can measure reporter dye contributions more accurately.
About the 5′ nuclease assay

The 5′ nuclease assay process (Figure 2 through Figure 5) takes place during PCR
amplification. This process occurs in every cycle and does not interfere with the
exponential accumulation of product.
Figure 1 Legend for Figures 2 through 5

NFQ = Nonfluorescent quencher
MGB = Minor groove binder
R = Reporter
P = Hot-start DNA polymerase
During PCR, the TaqMan MGB probe anneals specifically to a complementary

sequence between the forward and reverse primer sites (Figure 2).
When the probe is intact (Figure 2 and Figure 3), the proximity of the reporter dye to
the quencher dye results in suppression of the reporter fluorescence, primarily by
Förster-type energy transfer (Förster, 1948; Lakowicz, 1983).
Figure 2 Polymerization
Forward TaqMan
R NFQ MGB
Primer MGB probe
5′ P 3′
3′ 5′
5′ 3′
P Reverse
5′
Primer

Appendix D Background Information
About TaqMan® chemistry
Figure 3 Strand displacement
R
Forward TaqMan
NFQ MGB
Primer MGB probe
5′ P 3′
3′ 5′
5′ 3′
P Reverse
5′
Primer
The DNA polymerase cleaves only probes that are hybridized to the target (Figure 4).
Cleavage separates the reporter dye from the quencher dye; the separation of the
reporter dye from the quencher dye results in increased fluorescence by the reporter.
The increase in fluorescence occurs only if the target sequence is complementary to the
probe and is amplified during PCR. Because of these requirements, nonspecific
amplification is not detected.
Figure 4 Cleavage
R
Forward TaqMan
NFQ MGB
Primer MGB probe
5′ P 3′
3′ 5′
5′ 3′
P Reverse
5′
Primer
Polymerization of the strand continues, but because the 3′ end of the probe is blocked,
no extension of the probe occurs during PCR (Figure 5).
Figure 5 Completion of polymerization

TaqMan
R NFQ MGB
Forward MGB probe
Primer 3′
5′
3′ 5′
5′ 3′
5′
Reverse
Primer

About the assay information file (AIF)

The assay information file (AIF) contains reference information about your order and
technical details of all assays in the shipment. The AIF is included on the Information
CD accompanying your order, in a folder labeled with the Rack or Plate ID.
AIF formats
The AIF may be provided in TXT format and/or in both XML and HTML formats,
depending on the product line and order date. You can use the:
• HTML-format AIFs as a reference; open them in a Web browser.
• XML- and TXT-format AIFs for electronic data importation and manipulation.
Table 14 Assay information file formats and naming conventions
File format Filename convention (see Table 16)
HTML Assay_Info_ProdLine_SalesOrder_XXXX_RackID_YYYY
XML Assay_Info_ProdLine_SalesOrder_XXXX_RackID_YYYY
TXT Assay_Info_ProdLine_SalesOrder_XXXX_RackID_YYYY
-or-
ProdNum_LotNum_AIF
Table 15 Filename variables
Filename
Description
variable
LotNum The lot number of the assay (more than one lot number may be associated
with one manufacturing production number).
ProdLine The TaqMan assay product line:
• TaqMan Gene Expression Assays (TaqMan_GEx)
• Custom TaqMan Gene Expression Assays (Custom_TaqMan_GEx)
• Custom Plus TaqMan RNA Assays (Custom_TaqMan_ncRNA)
• TaqMan Non-coding RNA Assays (TaqMan_ncRNA)
ProdNum The manufacturing production number.
XXXX... A unique number assigned to the sales order.
YYYY... A unique, barcoded number located on various pieces of collateral; the
preceding term in the filename may be Rack ID, Plate ID, or Shipping Rack.

AIF field descriptions

Select assay information field descriptions are found in Understanding Your Shipment
(included on the CD with TaqMan Non-coding RNA Assays and certain TaqMan Gene
Expression Assays).
Assay information field descriptions for TaqMan Gene Expression Assays are listed in
Table 16, in the order in which they appear in the AIF.
Table 16 Assay information field descriptions for TaqMan® Gene Expression Assays
Examples by product type
Field name Description of content TaqMan® Gene Custom TaqMan®

Expression Gene Expression
Assays Assays
Customer Name Your organization or institution Company XYZ Company XYZ

(Sales) Order Number A unique number that identifies the 1234567890 1234567890
Applied Biosystems sales order
Ship Date The date when the assay was packaged for 7-Nov-2008 7-Nov-2008
shipment
Delivery Number A unique bar code number that identifies the 880309546 880309546
(Shipment ID) shipment
Note: The shipment ID also appears in
the plate ID.
Part Number A number that identifies the product line 4351372 4331348
Product Type The Applied Biosystems product line associated TaqMan® Gene Custom TaqMan®
with the assay Expression Assays Gene Expression
Assays
Assay ID An alphanumeric string that identifies the assay Rn01648213_m1 AIT9JN3
Lot Number A unique identifier assigned to each 080227T100615 080227T100615
manufacturing lot
Shipping Rack or The type of container in which the assay is 96-position tube 96-position tube rack
Plate Type shipped (such as a 96-position or a 16-position rack v1 v1
tube rack)
Shipping Rack, A bar code number on the label of each shipped 880309546-1 880309546-1
Plate ID, or Rack ID rack or plate
Vial/Tube Type The type of vial or tube that contains the assay 2D barcode 2D barcode labeled
labeled tube tube
Vial/Tube ID A unique, 10-digit bar code number on the 0004696076 0004696076
bottom of each assay vial or tube that identifies it
Well Location on the The location of the assay on the associated B02 B02
Shipping Rack or Plate shipping rack or plate
Assay Mix The concentration of the assay, including both 20✕ 20✕
Concentration primers and probe
Forward Primer Name Format: customer-designated sequence name – MYSEQ_F
(“MYSEQ”)_F


Assays Assays
Forward Primer The nucleotide sequence of the forward primer – GGACTTGCACGACTAA

Sequence
Forward Primer The concentration of the forward primer (µM) 18 18
Concentration
Reverse Primer Name Format: customer-designated sequence name – MYSEQ_R
(“MYSEQ”)_R
Reverse Primer The nucleotide sequence of the reverse primer – CCGTACGTCAATTGAC
Sequence
Reverse Primer The concentration of the reverse primer (µM) 18 18
Concentration
Reporter 1 Name Reporter 1 refers to the oligonucleotide probe. – MYSEQ_M
Format: customer-designated sequence name
(“MYSEQ”)_M.
Reporter 1 Dye The reporter dye label for the reporter 1 probe FAM™ FAM
Reporter 1 Sequence The nucleotide sequence of the reporter 1 probe – TTCGAACTGATCAT
Reporter 1 The concentration of the reporter 1 probe (µM) 5 5
Concentration
Reporter 1 Quencher The quencher used for the reporter 1 probe (for NFQ NFQ
example, Non-fluorescent Quencher [NFQ])
Reporter 2 Name Not applicable to TaqMan® Gene Expression – –
Assays
Reporter 2 Dye
Reporter 2 Sequence
Reporter 2
Concentration
Reporter 2 Quencher
Context Sequence The 25-nucleotide sequence surrounding the ...NNNNNNNNN... –
probe
Design Strand Not applicable to TaqMan® Gene Expression – –
Assays
Category The Celera Panther Protein Classification receptor –
(Level 1) for the gene product
Category ID A unique, 10-character alphanumeric REC1010000 –
abbreviation of the Panther category
classification
Group The Celera Panther Protein Classification protein kinase –
(Level 2) for the gene product receptor
Group ID A unique, 10-character alphanumeric 1010200000 –
abbreviation of the Panther group classification
Gene Symbol The Entrez Gene symbol for the gene SLC25A14 –


Assays Assays
Gene Name The Entrez Gene name for the gene solute carrier –
family 25
(mitochondrial
carrier, brain),
member 14
Chromosome The chromosome containing the gene 9 –
Species The organism for which the assay was designed Homo_sapiens –
Target Exon(s) The exon(s) that are spanned by the probe 2 –
NCBI Gene Reference The NCBI transcript identification number(s) NM_001735 –
corresponding to the assay target
NCBI SNP Reference Not applicable to TaqMan® Gene Expression – –
Assays
Medline Reference PubMed references for the gene – –
Celera ID (TaqMan Gene Expression Assays) The unique hCT11720402 –
Celera Discovery System (CDS) assay
- or -
identification number for the gene
Assay Name
(Custom TaqMan Gene Expression Assays) The – MYSEQ
customer-designated name of the sequence
used for custom assay design (such as
“MYSEQ”).
Cytogenetic Band The chromosomal band where the gene is 9q34 –
located.
If unavailable, then the chromosome number is
provided.


Assays Assays
SNP Type Not applicable to TaqMan® Gene Expression – –

Assay
Minor Allele
Frequency - Caucasian
Minor Allele
Frequency -African-
American
Minor Allele
Frequency -Japanese
Minor Allele
Frequency -Chinese
Celera Assembly Build
Number
Location on Celera
Assembly
NCBI Assembly Build
Number
Location on NCBI
Assembly


APPENDIX E
Safety
This appendix covers:

■ Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
General chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Chemical waste safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Biological hazard safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
■ General safety alerts for all chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Appendix E Safety
Chemical safety
Chemical safety
General chemical safety
Chemical hazard WARNING! CHEMICAL HAZARD. Before handling any chemicals, refer to
warning the Safety Data Sheet (SDS) provided by the manufacturer, and observe all
relevant precautions.
WARNING! CHEMICAL HAZARD. All chemicals in the instrument, including

liquid in the lines, are potentially hazardous. Always determine what chemicals
have been used in the instrument before changing reagents or instrument
components. Wear appropriate eyewear, protective clothing, and gloves when
working on the instrument.
WARNING! CHEMICAL HAZARD. Four-liter reagent and waste bottles can

crack and leak. Each 4-liter bottle should be secured in a low-density
polyethylene safety container with the cover fastened and the handles locked in
the upright position. Wear appropriate eyewear, clothing, and gloves when
handling reagent and waste bottles.
WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in

a glass container because of the risk of breaking or shattering. Reagent and
waste bottles can crack and leak. Each waste bottle should be secured in a low-
density polyethylene safety container with the cover fastened and the handles
locked in the upright position. Wear appropriate eyewear, clothing, and gloves
when handling reagent and waste bottles.
Chemical safety To minimize the hazards of chemicals:

guidelines • Read and understand the Safety Data Sheets (SDSs) provided by the chemical
manufacturer before you store, handle, or work with any chemicals or hazardous
materials. (See “About SDSs” on page 61.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the SDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use
only with adequate ventilation (for example, fume hood). For additional safety
guidelines, consult the SDS.
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
manufacturer’s cleanup procedures as recommended in the SDS.
• Comply with all local, state/provincial, or national laws and regulations related to
chemical storage, handling, and disposal.

Appendix E Safety
Chemical safety
SDSs
About SDSs Chemical manufacturers supply current Safety Data Sheets (SDSs) with shipments of
hazardous chemicals to new customers. They also provide SDSs with the first
shipment of a hazardous chemical to a customer after an SDS has been updated. SDSs
provide the safety information you need to store, handle, transport, and dispose of the
chemicals safely.
Each time you receive a new SDS packaged with a hazardous chemical, be sure to
replace the appropriate SDS in your files.
Obtaining The SDS for any chemical supplied by Applied Biosystems is available to you free
SDSs 24 hours a day. To obtain SDSs:
1. Go to www.appliedbiosystems.com, click Support, then select SDS.
2. In the Keyword Search field, enter the chemical name, product name, SDS part
number, or other information that appears in the SDS of interest. Select the
language of your choice, then click Search.
3. Find the document of interest, right-click the document title, then select any of the
following:
• Open – To view the document
• Print Target – To print the document
• Save Target As – To download a PDF version of the document to a
destination that you choose
Note: For the SDSs of chemicals not distributed by Applied Biosystems, contact
the chemical manufacturer.
Chemical waste safety
Chemical waste CAUTION! HAZARDOUS WASTE. Refer to Material Safety Data Sheets and
hazards local regulations for handling and disposal.
WARNING! CHEMICAL WASTE HAZARD. Wastes produced by Applied

Biosystems instruments are potentially hazardous and can cause injury, illness,
or death.
WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in

a glass container because of the risk of breaking or shattering. Reagent and
waste bottles can crack and leak. Each waste bottle should be secured in a low-
density polyethylene safety container with the cover fastened and the handles
locked in the upright position. Wear appropriate eyewear, clothing, and gloves
when handling reagent and waste bottles.

Appendix E Safety
Chemical safety
Chemical waste To minimize the hazards of chemical waste:

safety guidelines • Read and understand the Safety Data Sheets (SDSs) provided by the
manufacturers of the chemicals in the waste container before you store, handle, or
dispose of chemical waste.
• Provide primary and secondary waste containers. (A primary waste container
holds the immediate waste. A secondary container contains spills or leaks from
the primary container. Both containers must be compatible with the waste
material and meet federal, state, and local requirements for container storage.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the SDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use
only with adequate ventilation (for example, fume hood). For additional safety
guidelines, consult the SDS.
• Handle chemical wastes in a fume hood.
• After emptying a waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bottle in accordance with
good laboratory practices and local, state/provincial, or national environmental
and health regulations.
Waste disposal If potentially hazardous waste is generated when you operate the instrument, you
must:
• Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument 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 E Safety
General safety alerts for all chemicals
Biological hazard safety

General biohazard WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,
infectious agents, and blood of humans and other animals have the potential to
transmit infectious diseases. Follow all applicable local, state/provincial, and/or
national regulations. Wear appropriate protective equipment, which includes
but is not limited to: protective eyewear, face shield, clothing/lab coat, and
gloves. All work should be conducted in properly equipped facilities using the
appropriate safety equipment (for example, physical containment devices).
Individuals should be trained according to applicable regulatory and company/
institution requirements before working with potentially infectious materials.
Read and follow the applicable guidelines and/or regulatory requirements in
the following:
• U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories (www.cdc.gov/od/ohs/
biosfty/bmbl5/bmbl5toc.htm)
• Occupational Safety and Health Standards, Bloodborne Pathogens (29
CFR§1910.1030; www.access.gpo.gov/ nara/cfr/waisidx_01/
29cfr1910a_01.html).
• Your company’s/institution’s Biosafety Program protocols for working with/
handling potentially infectious materials.
Additional information about biohazard guidelines is available at:
www.cdc.gov

Avoid contact with skin, eyes, and/or clothing. Read the SDS, and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and gloves.

Appendix E Safety

Bibliography
Afonina, I., Zivarts, M., Kutyavin, I., et al., 1997. Efficient priming of PCR with short
oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25:2657–
2660.
Förster, V. T. 1948. Zwischenmolekulare Energiewanderung und Fluoreszenz. Annals of
Physics (Leipzig) 2:55–75.
Kutyavin, I.V., Lukhtanov, E.A., Gamper, H.B., and Meyer, R.B. 1997. Oligonucleotides
with conjugated dihydropyrroloindole tripeptides: base composition and backbone
effects on hybridization. Nucleic Acids Res. 25:3718–3723.
Lakowicz, J.R. 1983. Energy Transfer. In Principles of Fluorescence Spectroscopy, New York:
Plenum Press 303–339.
Longo, M.C., Berninger, M.S., and Hartley, J.L. 1990. Use of uracil DNA glycosylase to
control carryover contamination in polymerase chain reactions. Gene 93:125–128.
Livak, K.J. and Schmittgen, T.D. Analyzing real-time PCR data by the comparative
CT method. Nature Protocols 3, 1101-1108 (2008).
Livak, K.J. and Schmittgen, T.D. Analysis of relative gene expression data using
real-time, quantitative PCR and the 2-ΔΔ CT Method. Methods 25, 402–408 (2001).

Bibliography

Documentation and Support
Related documentation
Real-time PCR system Document PN/SN
All real-time PCR Custom TaqMan® Assays: Design and Ordering Guide 4367671
systems
Online Ordering Guide for TaqMan® Gene Expression Assays 127MI07-05
Online Selection Guide for TaqMan® Gene Expression Assays 127GU08-01
TaqMan® Gene Expression Assays Application Note: Amplification Efficiency of 127AP05-03
TaqMan® Gene Expression Assays Application Note: Using TaqMan® 127AP08-01
Endogenous Control Assays to Select an Endogenous Control for Experimental
Studies
Real-Time PCR Systems Chemistry Guide 4348358
High-Capacity cDNA Reverse Transcription Protocol 4375575
TaqMan® Gene Expression Master Mix Protocol 4371135
TaqMan® Universal PCR Master Mix (2✕) Protocol 4304449
TaqMan® Fast Advanced Master Mix Protocol 4444605
TaqMan® RNA-to-CT™ 1-Step Kit Protocol 4393463
White Paper: The Design Process for a New Generation of Quantitative Gene 127WP02-02
Expression Analysis Tools: TaqMan® Probe-Based Assays for Human, Mouse,
and Rat Genes
White Paper: Product Stability Study: TaqMan® Gene Expression Assays 127WP03-01
White Paper: TaqMan® Gene Expression Assays for Validating Hits from 127WP01-02
Fluorescent Microarrays
7900HT Fast system Performing Fast Gene Quantification: Quick Reference Card 4351892
Fast or standard sample
Relative Quantitation Using Comparative CT: Getting Started Guide 4364016
blocks
Performing Fast Gene Quantitation with 384-Well Plates: User Bulletin 4369584
7300, 7500, and Relative Quantification: Getting Started Guide 4347828
7500 Fast systems
ABI PRISM 7700 Relative Quantitation of Gene Expression: User Bulletin 4303859
Sequence Detection
System
StepOne™ and Reagent Guide 4379704
StepOnePlus™ systems
Relative Standard Curve and Comparative CT Experiments: Getting Started 4376785
Guide

Documentation and Support
Related documentation
Portable document format (PDF) versions of this and other documents are also
available on the TaqMan Gene Expression Assays CD.
Note: To open the user documentation included on the TaqMan® Gene
Expression Assays CD, use the Adobe® Acrobat® Reader® software available
from www.adobe.com
Note: For additional documentation, see “Obtaining support”.
Obtaining support
For the latest services and support information for all locations, go to:
www.appliedbiosystems.com
At the Applied Biosystems web site, you can:
• Access worldwide telephone and fax numbers to contact Applied Biosystems
Technical Support and Sales facilities.
• Search through frequently asked questions (FAQs).
• Submit a question directly to Technical Support.
• Order Applied Biosystems user documents, SDSs, certificates of analysis, and
other related documents.
• Download PDF documents.
• Obtain information about customer training.
• Download software updates and patches.

Part Number 4333458 Rev. N 11/2010
Headquarters Technical Resources and Support

5791 Van Allen Way | Carlsbad, CA 92008 USA For the latest technical resources and support information
Phone 760.603.7200 for all locations, please refer to our Web site at
www.lifetechnologies.com www.appliedbiosystems.com

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About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

PROTOCOL TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

TaqMan® Gene Expression Assays Protocol 3

APPENDIX A How to Order TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . 31

APPENDIX B Good PCR Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

APPENDIX C Duplex Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

APPENDIX D Background Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

4 TaqMan® Gene Expression Assays Protocol

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

TaqMan® Gene Expression Assays Protocol 5

6 TaqMan® Gene Expression Assays Protocol

Safety alert words

CAUTION! – Indicates a potentially hazardous situation that, if not avoided,

WARNING! – Indicates a potentially hazardous situation that, if not avoided,

DANGER! – Indicates an imminently hazardous situation that, if not avoided,

TaqMan® Gene Expression Assays Protocol 7

8 TaqMan® Gene Expression Assays Protocol

TaqMan® Gene Expression Assays

TaqMan® Gene Expression Assays Protocol 9

Table 1 TaqMan® Assay formulations

Product Type 20-µL rxns. Part number Availability

TaqMan® Gene Extra 75 4453320 Inventoried (20X)

10 TaqMan® Gene Expression Assays Protocol

Product Type 20-µL rxns. Part number Availability

TaqMan® Non- Small 360 4426961 Made-to-Order (20✕)

Storage and dilution

TaqMan® Gene Expression Assays Protocol 11

Materials and equipment not included

Materials for reverse transcription and PCR

Table 2 Required materials and equipment

Reverse transcription reagents Applied Biosystems

12 TaqMan® Gene Expression Assays Protocol

Compatible real-time instruments

TaqMan® Gene Expression Assays Protocol 13

Prepare the cDNA sample

Prepare the total RNA

Perform reverse transcription

Evaluate the cDNA GeneAm

Prepare the reaction mix and load the plate

Prepare the PCR reaction mix