High-Capacity cDNA Reverse Transcription Kits Protocol (PN 4375575)

High Capacity cDNA

Reverse Transcription Kits

For 200 and 1000 Reactions

Protocol

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

Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that

may appear in this document.

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IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR

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INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

DISCLAIMER OF LICENSE:

No license is conveyed with the purchase of this product under U.S. Patents, and corresponding patent claims outside the

U.S., relating to the 5′ nuclease and dsDNA-binding dye processes. Further information on purchasing licenses may be

obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California

94404, USA.

TRADEMARKS:

AB (Design), Applied Biosystems, and GeneAmp are registered trademarks and Applera, MicroAmp, and MultiScribe are

trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries.

TaqMan is a registered tradema rk of Roche Molecular Systems, Inc.

All other trademarks are the sole property of their respective owners.

Part Number 4375575 Rev. E

06/2010

iiiHigh Capacity cDNA Reverse Transcription Kits Protocol

Contents Front Matter

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Safety Alert Words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Chemical Hazard Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Chemical Safety Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

About MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

Obtaining MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

Chemical Waste Hazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Chemical Waste Safety Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . viii

Waste Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

Biological Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

High Capacity cDNA Reverse Transcription Kits Protocol . 1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Purpose of the High Capacity cDNA Reverse Transcription Kits . . . . 1

Kit Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Output Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

About This Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Good Laboratory Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

PCR Good Laboratory Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Materials and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Kit Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Kit Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Equipment Required but Not Supplied . . . . . . . . . . . . . . . . . . . . . . . 4

Materials Required but Not Supplied . . . . . . . . . . . . . . . . . . . . . . . . . 4

Using the High Capacity cDNA Reverse Transcription Kits . . . . . . . . . . . 6

Contents

High Capacity cDNA Reverse Transcription Kits Protocol

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

RNA Template Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Input Amount of Total RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Preparing the 2✕ Reverse Transcription Master Mix . . . . . . . . . . . . . 7

Preparing the cDNA Reverse Transcription Reactions . . . . . . . . . . . 8

Performing Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Storing cDNA Reverse Transcription Reactions . . . . . . . . . . . . . . . . 9

Appendix A, Examples of cDNA Yields from

Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Quantitative PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Yields for Different Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Yield vs. Reaction Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

vHigh Capacity cDNA Reverse Transcription Kits Protocol

Preface

This preface covers:

Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

Safety

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.

Definitions

IMPORTANT!

– Indicates information that is necessary for proper

instrument operation, accurate chemistry kit use, or safe use of a

chemical.

– 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.

– Indicates a potentially hazardous situation that,

if not avoided, could result in death or serious injury.

– 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.

Chemical Hazard

Warning

CHEMICAL HAZARD. Some of the chemicals

used with Applied Biosystems instruments and protocols are

potentially hazardous and can cause injury, illness, or death.

Preface

High Capacity cDNA Reverse Transcription Kits Protocol

Chemical Safety

Guidelines

To minimize the hazards of chemicals:

• Read and understand the Material Safety Data Sheets (MSDSs)

provided by the chemical manufacturer before you store, handle,

or work with any chemicals or hazardous materials. (See “About

MSDSs” on page vi.)

• 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 MSDS.

• 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 MSDS.

• Check regularly for chemical leaks or spills. If a leak or spill

occurs, follow the manufacturer’s cleanup procedures as

recommended in the MSDS.

• Comply with all local, state/provincial, or national laws and

regulations related to chemical storage, handling, and disposal.

About MSDSs

Chemical manufacturers supply current Material Safety Data Sheets

(MSDSs) with shipments of hazardous chemicals to new customers.

They also provide MSDSs with the first shipment of a hazardous

chemical to a customer after an MSDS has been updated. MSDSs

provide the safety information you need to store, handle, transport,

and dispose of the chemicals safely.

Each time you receive a new MSDS packaged with a hazardous

chemical, be sure to replace the appropriate MSDS in your files.

Obtaining

MSDSs

The MSDS for any chemical supplied by Applied Biosystems is

available to you free 24 hours a day. To obtain MSDSs:

1. Go to https://docs.appliedbiosystems.com/msdssearch.html

2. In the Search field of the MSDS Search page:

a. Type in the chemical name, part number, or other

information that you expect to appear in the MSDS of

interest.

b. Select the language of your choice.

c. Click Search.

vii

Safety

High Capacity cDNA Reverse Transcription Kits Protocol

3. To view, download, or print the document of interest:

a. Right-click the document title.

b. Select:

• Open – To view the document

• Save Target As – To download a PDF version of the

document to a destination that you choose

• Print Target – To print the document

4. To have a copy of an MSDS sent by fax or e-mail, in the Search

Results page:

a. Select Fax or Email below the document title.

b. Click RETRIEVE DOCUMENTS at the end of the

document list.

c. Enter the required information.

d. Click View/Deliver Selected Documents Now.

Note: For the MSDSs of chemicals not distributed by Applied

Biosystems, contact the chemical manufacturer.

Chemical Waste

Hazards

HAZARDOUS WASTE. Refer to Material

Safety Data Sheets and local regulations for handling and disposal.

CHEMICAL WASTE HAZARD. Wastes

produced by Applied Biosystems instruments are potentially

hazardous and can cause injury, illness, or death.

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.

viii

Preface

High Capacity cDNA Reverse Transcription Kits Protocol

Chemical Waste

Safety Guidelines

To minimize the hazards of chemical waste:

• Read and understand the Material Safety Data Sheets (MSDSs)

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 MSDS.

• 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 MSDS.

• Handle chemical wastes in a fume hood.

• After emptying the 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.

Safety

High Capacity cDNA Reverse Transcription Kits Protocol

Biological Hazard

Safety

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

(stock no. 017-040-00547-4; http://bmbl.od.nih.gov)

• Occupational Safety and Health Standards, Bloodborne

Pathogens (29 CFR§1910.1030; http://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:

http://www.cdc.gov

Preface

High Capacity cDNA Reverse Transcription Kits Protocol

How to Obtain Support

For the latest services and support information for all locations, go to

http://www.appliedbiosystems.com, then click the link for

Support.

At the Support page, you can:

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support

• Order Applied Biosystems user documents, MSDSs, certificates

of analysis, and other related documents

• Download PDF documents

• Obtain information about customer training

• Download software updates and patches

In addition, the Support page provides access to worldwide telephone

and fax numbers to contact Applied Biosystems Technical Support

and Sales facilities.

How to Obtain Support

High Capacity cDNA Reverse Transcription Kits Protocol

1High Capacity cDNA Reverse Transcription Kits Protocol

High Capacity cDNA Reverse

Transcription Kits Protocol

Overview

Purpose of the

High Capacity

cDNA Reverse

Transcription Kits

The High Capacity cDNA Reverse Transcription Kits for 200 and

1000 reactions contain all the reagents needed for reverse

transcription (RT) of total RNA to single-stranded cDNA using a

reaction size of 20 µL.

Kit Features

Use the kit for:

• Quantitatively converting up to 2 µg (for a 20-µL reaction) of

total RNA to cDNA

• Generating single-stranded cDNA suitable for quantitative PCR

applications

• Generating single-stranded cDNA suitable for short- or

long-term storage

Output

Applications

The cDNA reactions prepared using the High Capacity cDNA

Reverse Transcription Kits can be used in a variety of applications,

including:

• Quantitative PCR

• Archival storage

• Conversion to cRNA

About This

Protocol

This protocol describes:

• Procedures for using the kits

• Recommendations for using the cDNA created using the kits

• Examples of cDNA conversion performance obtained using

the kits

Good Laboratory Practices

High Capacity cDNA Reverse Transcription Kits Protocol

Good Laboratory Practices

PCR Good

Laboratory

Practices

PCR assays require special laboratory practices to avoid false

positive amplifications (Kwok and Higuchi, 1989). The high

throughput and repetition of these assays can lead to amplification of

a single DNA molecule (Saiki et al., 1985; Mullis and Faloona,

1987).

• Wear a clean lab coat (not previously worn while handling

amplified PCR products or used during sample preparation) and

clean gloves when preparing samples for PCR amplification.

• Change gloves whenever you suspect that they are

contaminated.

• Maintain separate areas, dedicated equipment, and supplies for:

– Sample preparation and PCR setup

– PCR amplification and post-PCR analysis

• Never bring amplified PCR products into the PCR setup area.

• Open and close all sample tubes and reaction plates carefully.

Do not splash or spray PCR samples.

• Keep reactions and components sealed as much as possible.

• Use positive-displacement pipettes or aerosol-resistant pipette

tips.

• Clean lab benches and equipment periodically with freshly

diluted 10% chlorine bleach solution.

Bibliography

Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR.

Nature 339:237-238.

Mullis, K.B. and Faloona, F.A. 1987. Specific synthesis of DNA in

vitro via a polymerase-catalyzed chain reaction. Methods Enzymol.

155:335-350.

Saiki, R.K., Scharf, S., Faloona,F., et al., 1985. Enzymatic

amplification of β-globin genomic sequences and restriction site

analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.

Materials and Equipment

High Capacity cDNA Reverse Transcription Kits Protocol

Materials and Equipment

Kit Types

Table 1 lists the four available kit sizes.

Kit Components

Table 2 lists all the kit components.

Kit Storage

Store all kit components at –15 to –25 °C.

Table 1 Available kits

Kit Name

Applied Biosystems

Part Number

High Capacity cDNA Reverse Transcription Kit,

1000 reactions

4368813

High Capacity cDNA Reverse Transcription Kit,

200 reactions

4368814

High Capacity cDNA Reverse Transcription Kit

with RNase Inhibitor, 1000 reactions

4374967

High Capacity cDNA Reverse Transcription Kit

with RNase Inhibitor, 200 reactions

4374966

Table 2 Kit components

Component

Quantity in

1000 reaction kit

Quantity in

200 reaction kit

10✕ RT Buffer, 1.0 mL 2 tubes 1 tube

10✕ RT Random Primers, 1.0 mL 2 tubes 1 tube

25✕ dNTP Mix (100 mM) 1 tube, 1.0 mL 1 tube, 0.2 mL

MultiScribe

™

Reverse

Transcriptase, 50 U/µL

1 tube, 1.0 mL 2 tubes, 0.1 mL

RNase Inhibitor

‡

, 100 µL

‡ Only in orders that include RNase inhibitor (PN 4374966 or 4374967).

10 tubes 2 tubes

Materials and Equipment

High Capacity cDNA Reverse Transcription Kits Protocol

Equipment

Required but

Not Supplied

Table 3 lists the equipment required to use the High Capacity cDNA

Reverse Transcription Kits.

Materials

Required but

Not Supplied

Table 4 lists the non-kit materials required to use the High Capacity

cDNA Reverse Transcription Kits.

Table 3 Required equipment

Equipment Source

Thermal cycler (one of the following):

• Applied Biosystems 9800 Fast

Thermal Cycler with 96-Well Aluminum

Sample Block Module

• GeneAmp

PCR System 9700

Thermal Cycler

• GeneAmp

PCR System 9600

Thermal Cycler

Applied Biosystems

(PN 4352604)

See your

Applied Biosystems

sales representative

Centrifuge with 96-well adapter Major laboratory supplier

(MLS)

Microcentrifuge MLS

Vortexer MLS

Table 4 Required materials

Materials/Consumables Source

MicroAmp

™

Fast 96-Well Reaction Plate Applied Biosystems

(PN 4346907)

MicroAmp

™

Fast 96-Well Reaction Plate

with Bar Code

Applied Biosystems

(PN 4346906)

MicroAmp

™

Optical 96-Well Reaction Plate Applied Biosystems

(PN N8010560)

MicroAmp

™

8-tube strip Applied Biosystems

(PN N8010580)

MicroAmp

™

Fast 8-tube strip Applied Biosystems

(PN 4358203)

MicroAmp

™

8-cap strip Applied Biosystems

(PN N8010535)

Materials and Equipment

High Capacity cDNA Reverse Transcription Kits Protocol

Clear adhesive film Applied Biosystems

(PN 4306311)

Optical adhesive film Applied Biosystems

(PN 4360954)

MicroAmp

™

Optical 96-Well Reaction Plates

and Optical Caps

Applied Biosystems

(PN 403012)

MicroAmp

™

Optical Caps, 8 Caps/Strip Applied Biosystems

(PN 4323032)

Reagent Tubes with Caps, 10-mL Applied Biosystems

(PN 4305932)

RNase Inhibitor

‡

(for kits PN 4368813 and 4368814)

Applied Biosystems

(PN N8080119)

Nuclease-free H

OMLS

Pipette tips, aerosol-resistant MLS

Pipettors, positive-displacement MLS

Disposable gloves MLS

Cap Installing Tool Applied Biosystems

(PN 4330015)

Adhesive Seal Applicator Applied Biosystems

(PN 4333183)

‡ Included with kits PN 4374966 and 4374967.

Table 4 Required materials (continued)

Materials/Consumables Source

Using the High Capacity cDNA Reverse Transcription Kits

High Capacity cDNA Reverse Transcription Kits Protocol

Using the High Capacity cDNA Reverse

Transcription Kits

Overview

To synthesize single-stranded cDNA from total RNA using the High

Capacity cDNA Reverse Transcription Kits:

RNA Template

Guidelines

For optimal performance of the High Capacity cDNA Reverse

Transcription Kits, Applied Biosystems recommends using RNA

that is:

• Free of inhibitors of reverse transcription and PCR

• Dissolved in PCR-compatible buffer or water

• Free of RNase activity

Note: If you suspect that the RNA contains RNase activity, add

RNase Inhibitor to the reverse transcription reaction at a final

concentration of 1.0 U/µL.

Input Amount of

Total RNA

Use up to 2 µg of total RNA per 20-µL reaction.

Add total RNA to the 2✕ RT Master Mix

to create a 1✕ mix

Use the reverse transcription

reactions (cDNA) directly for

uantitative or other PCR a

lications

Store the

reverse transcription

reactions

(

cDNA

)

Prepare the 2✕ Reverse Transcription

Master Mix

Perform reverse transcription

in a thermal cycler

Using the High Capacity cDNA Reverse Transcription Kits

High Capacity cDNA Reverse Transcription Kits Protocol

Preparing the

2✕ Reverse

Transcription

Master Mix

Prepare the 2✕ RT master mix using the kit components before

preparing the reaction plate.

To prepare the 2✕ RT master mix (per 20-µL reaction):

1. Allow the kit components to thaw on ice.

2. Referring to the table below, calculate the volume of

components needed to prepare the required number of

reactions.

Note: Prepare the RT master mix on ice.

IMPORTANT! Include additional reactions in the

calculations to provide excess volume for the loss that occurs

during reagent transfers.

CHEMICAL HAZARD. 10✕ Reverse

Transcription Buffer may cause eye, skin, and respiratory

tract irritation. Read the MSDS, and follow the handling

instructions. Wear appropriate eyewear, clothing, and gloves.

3. Place the 2✕ RT master mix on ice and mix gently.

Component

Volume/Reaction

(µL)

Kit with

RNase Inhibitor

Kit without

RNase Inhibitor

10✕ RT Buffer 2.0 2.0

25✕ dNTP Mix (100 mM) 0.8 0.8

10✕ RT Random Primers 2.0 2.0

MultiScribe

™

Reverse Transcriptase

1.0 1.0

RNase Inhibitor 1.0 —

Nuclease-free H

O3.24.2

Total per Reaction 10.0 10.0

Using the High Capacity cDNA Reverse Transcription Kits

High Capacity cDNA Reverse Transcription Kits Protocol

Preparing the

cDNA Reverse

Transcription

Reactions

Performing

Reverse

Transcription

To prepare the cDNA RT reactions:

1. Pipette 10 µL of 2✕ RT master mix into each well of a

96-well reaction plate or individual tube.

2. Pipette 10 µL of RNA sample into each well, pipetting up

and down two times to mix.

3. Seal the plates or tubes.

4. Briefly centrifuge the plate or tubes to spin down the

contents and to eliminate any air bubbles.

5. Place the plate or tubes on ice until you are ready to load the

thermal cycler.

To perform reverse transcription:

1. Program the thermal cycler conditions using one of the

thermal cyclers listed in Table 3 on page 4.

IMPORTANT! These conditions are optimized for use with

the High Capacity cDNA Reverse Transcription Kits.

2. Set the reaction volume to 20 µL.

3. Load the reactions into the thermal cycler.

4. Start the reverse transcription run.

Step 1 Step 2 Step 3 Step 4

Temperature (°C) 25 37 85 4

Time 10 min 120 min 5 min ∞

Using the High Capacity cDNA Reverse Transcription Kits

High Capacity cDNA Reverse Transcription Kits Protocol

Storing cDNA

Reverse

Transcription

Reactions

You can store cDNA RT plates or tubes prepared using the High

Capacity cDNA Reverse Transcription Kits for short-term or long-

term storage.

IMPORTANT! If required, briefly centrifuge the archive plates or

tubes before storing to spin down the contents and to eliminate any

air bubbles.

Storage Duration

Storage Temperature

(°C)

Short-term (up to 24 hours before use)

‡

‡ For prolonged storage at 2 to 6 °C, add EDTA to a final concentration of 1 mM to

chelate cations and to prevent nucleic acid degradation.

2 to 6

Long-term −15 to −25

Using the High Capacity cDNA Reverse Transcription Kits

High Capacity cDNA Reverse Transcription Kits Protocol

11High-Capacity cDNA Reverse Transcription Kits Protocol

Appendix A: Examples of cDNA Yields

from Reverse Transcription

Quantitative PCR

To determine the yield of the cDNA from the reverse transcription of

total RNA, use quantitative PCR to test various input amounts of

RNA for the cDNA yield of different gene targets.

The table below lists some targets and Applied Biosystems kits that

you can use to evaluate the yield of cDNA conversion.

You can use other TaqMan

Gene Expression Assays to evaluate the

yield of cDNA conversion. For a list of available assays, visit

www.appliedbiosystems.com.

Yields for

Different Targets

For the example in this appendix, the input total RNA was obtained

from human Raji cells, and the RNA was converted to cDNA using

the High-Capacity cDNA Reverse Transcription Kits.

Figure 1 shows an example of quantitative PCR results from the

cDNA for 11 different gene targets, which vary in expression levels.

Note: The amplicon for β

-microglobulin in this study was

specifically designed to be A/U rich.

The threshold cycle (C

) values are plotted against RNA input

amounts of 0.1, 1.0, and 10.0 µg in 100-µL reactions.

Gene

Tar ge t

Kit

Applied Biosystems

Part Number

18S TaqMan

Ribosomal RNA Control

Reagents

4308329

GAPDH TaqMan

GAPDH Control Reagents

[Human]

402869

GAPDH TaqMan

Rodent GAPDH Control

Reagents

4308313

β-actin TaqMan

β-actin Detection

Reagents

401846

Appendix A Examples of cDNA Yields from Reverse Transcription

High-Capacity cDNA Reverse Transcription Kits Protocol

Figure 1 The expected ∆C

values of 3.3 for each tenfold

increase in the RNA input quantity are obtained for 11 different

RNA transcripts converted to cDNA from different input quantities

of total RNA.

Yield vs.

Reaction Time

To achieve optimal conversion, Applied Biosystems recommends

allowing reverse transcription to occur for 120 minutes at 37 °C.

Figures 2, 3, and 4 show C

values plotted against reaction time in

minutes for three different targets (18S, GAPDH, and

-microglobulin) and three input amounts of RNA (0.1, 1.0, and

10.0 µg in 100-µL reactions).

C Value

RNA Input (µg)

0.1 1.0 10.0

18S

β-actin

β -microglobulin

cyclophilin

GAPDH

β-glucuronidase

hypoxanthine phosphoribosyl transferase

phosphoglycerate kinase

acidic ribosomal protein

TATA -binding protein

transferrin receptor

13High-Capacity cDNA Reverse Transcription Kits Protocol

Figure 2 The rate of conversion of 18S RNA to cDNA reaches a

maximum at 120 minutes with 10 µg of input RNA and at

30 minutes or less with 0.1 to 1.0 µg of input RNA (based on

100-µL reactions).

Figure 3 The rate of conversion of GAPDH RNA to cDNA reaches

a maximum at 60 minutes or less at all RNA input levels (based on

100-µL reactions).

Time (min)

C Value

10 30 50 70 90 110 130

14.0

16.0

18.0

20.0

22.0

10.0 µg

1.0 µg

0.1 µg

Time (min)

C Value

10 30 50 70 90 110 130

27.0

29.0

31.0

33.0

35.0

10.0 µg

1.0 µg

0.1 µg

Appendix A Examples of cDNA Yields from Reverse Transcription

High-Capacity cDNA Reverse Transcription Kits Protocol

Figure 4 The rate of conversion of β

-microglobulin RNA to cDNA

reaches a maximum at 60 minutes or less at all RNA input levels

(based on 100-µL reactions).

Time (min)

C Value

10 30 50 70 90 110 130

24.0

26.0

28.0

30.0

32.0

10.0 µg

1.0 µg

0.1 µg

15High Capacity cDNA Reverse Transcription Kits Protocol

Index

Numerics

2✕ reverse transcription (RT) master mix,

preparing 7

Applied Biosystems

contacting x

Technical Support x

biohazardous waste, handling viii

CAUTION, description v

cDNA reverse transcription (RT) reactions

output applications 1

preparing 8

storing 9

using 1

cDNA yields from reverse transcription,

examples 11

chemical safety vi

chemical waste

hazards vii

safety viii

DANGER, description v

equipment, required 4

guidelines

chemical safety vi

chemical waste disposal vii

chemical waste safety viii

RNA template 6

hazards, chemical waste vii

High-Capacity cDNA Reverse Transcription Kits

contents 3

storing 3

IMPORTANT, description v

kit. See High-Capacity cDNA Reverse

Transcription Kits.

master mix. See 2✕ reverse transcription (RT)

master mix.

materials, required 4

MSDSs

description vi

obtaining vi

, x

output applications for cDNA from RT

reactions 1

PCR good laboratory practices 2

performing reverse transcription 8

preparing

Index

High Capacity cDNA Reverse Transcription Kits Protocol

2✕ reverse transcription (RT) master mix 7

cDNA reverse transcription (RT) reactions 8

protocol workflow 6

quantitative PCR 11

radioactive waste, handling viii

required

equipment 4

materials 4

reverse transcription, performing 8

RNA template guidelines 6

RNA, total input amount 6

safety

biological hazards ix

chemical waste vii

guidelines vi

, vii, viii

storing

cDNA reverse transcription (RT) reactions 9

High-Capacity cDNA Reverse Transcription

Kits 3

Technical Support, contacting x

training, information on x

using cDNA reverse transcription (RT)

reactions 1

WARNING, description v

waste disposal, guidelines viii

workflow 6

yield of cDNA from RNA

yield vs. reaction time 12

yields for different targets 11

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06/2010

Part Number 4375575 Rev. E