QuantiTect® Reverse Transcription Handbook

QuantiTect

Reverse Transcription

Handbook

For cDNA synthesis with integrated removal of genomic

DNA contamination

For use in real-time two-step RT-PCR

March 2009

Sample & Assay Technologies

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QIAGEN Sample and Assay Technologies

QIAGEN is the leading provider of innovative sample and assay technologies, enabling

the isolation and detection of contents of any biological sample. Our advanced,

high-quality products and services ensure success from sample to result.

QIAGEN sets standards in:

■ Purification of DNA, RNA, and proteins

■ Nucleic acid and protein assays

■ microRNA research and RNAi

■ Automation of sample and assay technologies

Our mission is to enable you to achieve outstanding success and breakthroughs. For

more information, visit www.qiagen.com.

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QuantiTect Reverse Transcription Handbook 03/2009 3

Contents

Kit Contents 4

Shipping and Storage 4

Product Warranty and Satisfaction Guarantee 4

Product Use Limitations 5

Technical Assistance 5

Safety Information 5

Quality Control 6

Product Description 6

Introduction 7

Principle and procedure 7

Enzymatic activities of reverse transcriptase 9

Equipment and Reagents to Be Supplied by User 10

Protocol

■ Reverse Transcription with Elimination of Genomic DNA for Quantitative,

Real-Time PCR 11

Troubleshooting Guide 14

Appendix A: General Remarks on Handling RNA 18

Appendix B: Preparation, Storage, Quantification, and Determination

of Quality of RNA 20

Appendix C: Quantitative, Real-Time Two-Step RT-PCR 23

Appendix D: Recommended Controls for Quantitative, Real-Time RT-PCR 24

References 25

Ordering Information 26

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Kit Contents

QuantiTect Reverse Transcription Kit (10) (50) (200)

Catalog no. 205310 205311 205313

Number of standard reactions* 10 50 200

gDNA Wipeout Buffer, 7x 100 µl 100 µl 4 x 100 µl

Quantiscript

Reverse Transcriptase

†

10 µl 50 µl 4 x 50 µl

Quantiscript RT Buffer, 5x

‡

200 µl 200 µl 4 x 200 µl

RT Primer Mix 50 µl 50 µl 4 x 50 µl

RNase-Free Water 1.9 ml 1.9 ml 4 x 1.9 ml

Handbook 1 1 1

* A standard reaction is 20 µl in volume with 10 pg to 1 µg total RNA.

†

A mixture of the QIAGEN

products Omniscript

Reverse Transcriptase and Sensiscript

Reverse

Transcriptase. Also contains RNase inhibitor.

‡

Includes Mg

and dNTPs.

Shipping and Storage

The QuantiTect Reverse Transcription Kit is shipped on dry ice. The kit, including all

reagents and buffers, should be stored immediately upon receipt at –20°C in a constant-

temperature freezer.

Product Warranty and Satisfaction Guarantee

QIAGEN guarantees the performance of all products in the manner described in our

product literature. The purchaser must determine the suitability of the product for its

particular use. Should any product fail to perform satisfactorily due to any reason other

than misuse, QIAGEN will replace it free of charge or refund the purchase price. We

reserve the right to change, alter, or modify any product to enhance its performance

and design. If a QIAGEN product does not meet your expectations, simply call your

local Technical Service Department or distributor. We will credit your account or

exchange the product — as you wish. Separate conditions apply to QIAGEN scientific

instruments, service products, and to products shipped on dry ice. Please inquire for

more information.

A copy of QIAGEN terms and conditions can be obtained on request, and is also

provided on the back of our invoices. If you have questions about product specifications

or performance, please call QIAGEN Technical Services or your local distributor (see

back cover or visit www

.qiagen.com).

QuantiTect Reverse Transcription Handbook 03/20094

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QuantiTect Reverse Transcription Handbook 03/2009 5

Product Use Limitations

The QuantiTect Reverse Transcription Kit is intended for research use. No claim or

representation is intended to provide information for the diagnosis, prevention, or

treatment of a disease.

All due care and attention should be exercised in the handling of the products. We

recommend all users of QIAGEN products to adhere to the NIH guidelines that have

been developed for recombinant DNA experiments, or to other applicable guidelines.

Technical Assistance

At QIAGEN, we pride ourselves on the quality and availability of our technical support.

Our Technical Service Departments are staffed by experienced scientists with extensive

practical and theoretical expertise in sample and assay technologies and the use of

QIAGEN products. If you have any questions or experience any difficulties regarding

the QuantiTect Reverse Transcription Kit or QIAGEN products in general, please do not

hesitate to contact us.

QIAGEN customers are a major source of information regarding advanced or

specialized uses of our products. This information is helpful to other scientists as well as

to the researchers at QIAGEN. We therefore encourage you to contact us if you have

any suggestions about product performance or new applications and techniques.

For technical assistance and more information, please see our Technical Support

Center at www

.qiagen.com/Support or call one of the QIAGEN Technical Service

Departments or local distributors (see back cover or visit www

.qiagen.com).

Safety Information

When working with chemicals, always wear a suitable lab coat, disposable gloves,

and protective goggles. For more information, please consult the appropriate material

safety data sheets (MSDSs). These are available online in convenient and compact PDF

format at www

.qiagen.com/Support/MSDS.aspx where you can find, view, and print

the MSDS for each QIAGEN kit and kit component.

24-hour emergency information

Emergency medical information in English, French, and German can be obtained 24

hours a day from:

Poison Information Center Mainz, Germany

Tel: +49-6131-19240

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Quality Control

In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of

QuantiTect Reverse Transcription Kit is tested against predetermined specifications to

ensure consistent product quality.

Product Description

Component Description

gDNA Wipeout Buffer, 7x Buffer for effective elimination of genomic DNA

contamination from starting RNA samples.

Quantiscript Reverse Developed for use in real-time two-step RT-PCR.

Transcriptase Contains an optimized mixture of the QIAGEN products

Omniscript Reverse Transcriptase and Sensiscript Reverse

Transcriptase, which are recombinant heterodimeric

enzymes expressed in

E. coli

. Also contains RNase

inhibitor, a 50 kDa protein that strongly inhibits RNases A,

B, and C as well as human placental RNases.

Quantiscript RT Buffer, 5x Buffer optimized for reverse transcription with

Quantiscript Reverse Transcriptase; contains dNTPs.

RT Primer Mix Optimized blend of oligo-dT and random primers

dissolved in water. RT Primer Mix allows high cDNA

yields from all regions of RNA transcripts, even from

5' regions.

RNase-Free Water Ultrapure quality, PCR-grade

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QuantiTect Reverse Transcription Handbook 03/2009 7

Introduction

The QuantiTect Reverse Transcription Kit provides a fast and convenient procedure for

efficient reverse transcription and effective genomic DNA elimination. The kit is

dedicated for use in real-time two-step RT-PCR, and provides high cDNA yields for

sensitive quantification of even low-abundance transcripts.

Principle and procedure

The QuantiTect Reverse Transcription procedure takes only 20 minutes and comprises

2 main steps: elimination of genomic DNA and reverse transcription (see flowchart,

next page).

Elimination of genomic DNA

The purified RNA sample is briefly incubated in gDNA Wipeout Buffer at 42°C for

2 minutes to effectively remove contaminating genomic DNA. In contrast to other

methods, the RNA sample is then used directly in reverse transcription.

Accurate results in real-time RT-PCR depend on the use of primers or probes designed

to eliminate or minimize detection of genomic DNA. If such primers or probes are not

available, then genomic DNA contamination in RNA samples must be eliminated.

Reverse transcription

After genomic DNA elimination, the RNA sample is ready for reverse transcription using

a master mix prepared from Quantiscript Reverse Transcriptase, Quantiscript RT Buffer,

and RT Primer Mix. The entire reaction takes place at 42°C and is then inactivated at

95°C. In contrast to other methods, additional steps for RNA denaturation, primer

annealing, and RNase H digestion are not necessary.

Quantiscript Reverse Transcriptase has a high affinity for RNA and is optimized for

efficient and sensitive cDNA synthesis from 10 pg to 1 µg of RNA. This high RNA

affinity, in combination with Quantiscript RT Buffer, enables high cDNA yields, even

from templates with high GC-content or complex secondary structure.

RT Primer Mix ensures cDNA synthesis from all regions of RNA transcripts, even from

5' regions. This allows high yields of cDNA template for real-time PCR analysis

regardless of where the target region is located on the transcript.

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QuantiTect Reverse Transcription Handbook 03/20098

Mix RNA,

gDNA Wipeout Buffer,

and RNase-free water

Incubate at 42°C for 2 min

Add Quantiscript Reverse

Transcriptase, Quantiscript RT

Buffer, and RT Primer Mix, and mix

Add cDNA to real-time

PCR mix and distribute

Quantitative, real-time PCR

Incubate at 42°C for 15 min

Incubate at 95°C for 3 min to

inactivate Quantiscript Reverse

Transcriptase

QuantiTect Reverse Transcription Procedure

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QuantiTect Reverse Transcription Handbook 03/2009 9

Enzymatic activities of reverse transcriptase

Reverse transcriptase enzymes are generally derived from RNA-containing retroviruses

such as avian myeloblastosis virus (AMV), Moloney murine leukemia virus (MMLV), or

human immunodeficiency virus (HIV). Quantiscript Reverse Transcriptase is from a new

source.

Figure 1. cDNA synthesis. Quantiscript Reverse Transcriptase in first-strand cDNA synthesis.

In general, reverse transcriptase is a multifunctional enzyme with 3 distinct enzymatic

activities: an RNA-dependent DNA polymerase, a hybrid-dependent exoribonuclease

(RNase H), and a DNA-dependent DNA polymerase. In vivo, the combination of these

3 activities allows transcription of the single-stranded RNA genome into double-

stranded DNA for retroviral infection. For reverse transcription in vitro (Figure 1), the

first 2 activities are utilized to produce single-stranded cDNA:

■ RNA-dependent DNA-polymerase activity (reverse transcription) transcribes cDNA

from an RNA template. This activity of Quantiscript Reverse Transcriptase allows

synthesis of cDNA for use in quantitative, real-time PCR.

■ RNase H activity of Quantiscript Reverse Transcriptase specifically degrades only

the RNA in RNA:DNA hybrids. Therefore, this RNase H activity affects RNA

hybridized to cDNA, but has no effect on pure RNA. A separate RNA degradation

step using RNase H enzyme is not necessary prior to real-time PCR. Furthermore,

the Quantiscript RNase H activity, acting during reverse transcription, may

improve the sensitivity of subsequent real-time PCR.

mRNA

cDNA

AAAAAA

Primer annealing

Reverse transcription

(RNA-dependent DNA polymerase)

RNA degradation

(RNase H)

Quantitative, real-time PCR

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QuantiTect Reverse Transcription Handbook 03/200910

Equipment and Reagents to Be Supplied by User

When working with chemicals, always wear a suitable lab coat, disposable gloves,

and protective goggles. For more information, consult the appropriate material safety

data sheets (MSDSs), available from the product supplier.

For genomic DNA elimination and reverse transcription:

■ Plastic tubes (for 20 µl reactions)

■ Ice

■ Heating block or water bath (capable of reaching 95°C)

■ Vortexer

■ Microcentrifuge

■ Optional: gene-specific primers

For quantitative, real-time PCR:

■ Optimized kit for quantitative, real-time PCR, which includes

Taq

polymerase;

quantitative, real-time PCR buffer; primers; probe or SYBR

Green I dye; and

nucleotides (for details, see Appendix C, page 23).

■ QIAGEN offers optimized, ready-to-run kits for highly specific and sensitive real-

time PCR:

■ Rotor-Gene

Kits — for ultrafast results on the Rotor-Gene Q

■ QuantiFast

Kits — for fast cycling on instruments from other suppliers

■ QuantiTect Kits — for standard cycling on instruments from other suppliers;

includes optional UNG pretreatment

Kits are available for SYBR Green, probe, or multiplex detection. For more details,

visit www

.qiagen.com/PCR. For ordering information, see page 26.

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Protocol: Reverse Transcription with Elimination of

Genomic DNA for Quantitative, Real-Time PCR

Important points before starting

■ The protocol is optimized for use with 10 pg to 1 µg of RNA. If using >1 µg RNA,

scale up the reaction linearly to the appropriate volume.

■ Set up all reactions on ice to minimize the risk of RNA degradation.

■ RNase inhibitor and dNTPs are already included in the kit components. Do not add

additional RNase inhibitor and dNTPs.

■ RT Primer Mix (supplied) or gene-specific primers (not supplied) should be used.

RT Primer Mix is optimized to provide high cDNA yields for all regions of RNA

transcripts. If using gene-specific primers, we recommend using a final

concentration of 0.7 µM or testing a range of final concentrations from 0.5 µM

to 1 µM.

■ For convenience, premix RT Primer Mix and 5x Quantiscript RT Buffer in a 1:4 ratio

if RT Primer Mix will be used routinely for reverse transcription. This premix is

stable when stored at –20°C.

■ Separate denaturation and annealing steps are not necessary before starting the

reverse-transcription reaction.

■ If using a reaction volume of 200 µl or greater for reverse transcription, make sure

the reaction tube is efficiently heated (e.g., if using a heating block, carefully fill

each well with a drop of water so that heat can be efficiently transferred from the

block to the tube).

■ After reverse transcription, the reaction must be inactivated by incubation at 95°C

for 3 minutes.

■ If working with RNA for the first time, read Appendix A, page 18.

■ For details on performing real-time PCR after reverse transcription, see Appendix

C, page 23. For details on appropriate controls, see Appendix D, page 24.

■ Users of the FastLane

Cell cDNA Kit: If you have purchased the QuantiTect Reverse

Transcription Kit in order to perform additional reverse-transcription reactions with

the FastLane Cell cDNA Kit, follow the protocol in the

FastLane Cell cDNA

Handbook.

Do not follow the protocol in the

QuantiTect Reverse Transcription

Handbook.

Things to do before starting

■ Dissolve any precipitates in gDNA Wipeout Buffer by vortexing. If necessary,

briefly incubate the buffer at 37°C until the precipitates dissolve.

Protocol

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Procedure

1. Thaw template RNA on ice. Thaw gDNA Wipeout Buffer, Quantiscript Reverse

Transcriptase, Quantiscript RT Buffer, RT Primer Mix, and RNase-free water at room

temperature (15–25°C).

Mix each solution by flicking the tubes. Centrifuge briefly to collect residual liquid

from the sides of the tubes, and then store on ice.

2. Prepare the genomic DNA elimination reaction on ice according to Table 1.

Mix and then store on ice.

Note: If setting up more than one reaction, prepare a volume of master mix 10%

greater than that required for the total number of reactions to be performed. Then

distribute the appropriate volume of master mix into individual tubes followed by

each RNA sample. Keep the tubes on ice.

Note: The protocol is for use with 10 pg to 1 µg RNA. If using >1 µg RNA, scale

up the reaction linearly. For example, if using 2 µg RNA, double the volumes of

all reaction components for a final 28 µl reaction volume.

Table 1. Genomic DNA elimination reaction components

Component Volume/reaction Final concentration

gDNA Wipeout Buffer, 7x 2 µl 1x

Template RNA Variable (up to 1 µg*)

RNase-free water Variable

Total volume 14 µl –

* This amount corresponds to the entire amount of RNA present, including any rRNA, mRNA, viral RNA, and

carrier RNA present, and regardless of the primers used or cDNA analyzed.

3. Incubate for 2 min at 42°C. Then place immediately on ice.

Note: Do not incubate at 42°C for longer than 10 min.

4. Prepare the reverse-transcription master mix on ice according to Table 2.

Mix and then store on ice. The reverse-transcription master mix contains all

components required for first-strand cDNA synthesis except template RNA.

Note: If setting up more than one reaction, prepare a volume of master mix 10%

greater than that required for the total number of reactions to be performed.

Note: The protocol is for use with 10 pg to 1 µg RNA. If using >1 µg RNA, scale

up the reaction linearly. For example, if using 2 µg RNA, double the volumes of

all reaction components for a final 40 µl reaction volume.

Protocol

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Protocol

QuantiTect Reverse Transcription Handbook 03/2009 13

Table 2. Reverse-transcription reaction components

Component Volume/reaction Final concentration

Reverse-transcription master mix

Quantiscript Reverse Transcriptase* 1 µl

Quantiscript RT Buffer, 5x

†‡

4 µl 1x

RT Primer Mix

‡

1 µl

Template RNA

Entire genomic DNA 14 µl (add at step 5)

elimination reaction (step 3)

Total volume 20 µl –

* Also contains RNase inhibitor.

†

Includes Mg

and dNTPs.

‡

For convenience, premix RT Primer Mix and 5x Quantiscript RT Buffer in a 1:4 ratio if RT Primer Mix will be

used routinely for reverse transcription. This premix is stable when stored at –20°C. Use 5 µl of the premix

per 20 µl reaction.

5. Add template RNA from step 3 (14 µl) to each tube containing reverse-transcription

master mix.

Mix and then store on ice.

6. Incubate for 15 min at 42°C.

In some rare cases (e.g., if the RT-PCR product is longer than 200 bp or if analyzing

RNAs with a very high degree of secondary structure), increasing the incubation

time up to 30 min may increase cDNA yields.

7. Incubate for 3 min at 95°C to inactivate Quantiscript Reverse Transcriptase.

8. Add an aliquot of each finished reverse-transcription reaction to real-time PCR mix

(see Appendix C, page 23).

Store reverse-transcription reactions on ice and proceed directly with real-time

PCR, or for long-term storage, store reverse-transcription reactions at –20°C.

For real-time PCR, we recommend using a Rotor-Gene Kit, QuantiFast Kit, or

QuantiTect Kit (see page 10).

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QuantiTect Reverse Transcription Handbook 03/200914

Troubleshooting Guide

This troubleshooting guide may be helpful in solving any problems that may arise. For

more information, see also the Frequently Asked Questions page at our Technical

Support Center: www

.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN

Technical Services are always happy to answer any questions you may have about

either the information and protocol in this handbook or sample and assay technologies

(for contact information, see back cover or visit www

.qiagen.com).

Comments and suggestions

No product, or product detected late in real-time PCR (problems occurring during

reverse transcription)

a) Pipetting error or missing Check the pipets used for experimental setup.

reagent when setting up Mix all reagents well after thawing, and repeat

reverse-transcription reaction the reverse-transcription reaction.

b) Incorrect setup of Be sure to set up the reaction on ice.

reverse-transcription reaction

c) Volume of reverse-transcription Adding a high volume of reverse-transcription

reaction added to the reaction to the PCR mix may reduce amplification

real-time PCR was too high efficiency and the linearity of the reaction.

Generally, the volume of reverse-transcription

reaction added should not exceed 10% of the

final PCR volume.

d) Temperature of Reverse transcription should be carried out at

reverse-transcription reaction 42°C. Check the temperature of your heating

block or water bath. In rare cases, when

analyzing RNAs with a very high degree of

secondary structure, it may be advantageous to

increase the temperature up to 50°C. However,

temperatures >42°C will reduce the activity of

Quantiscript Reverse Transcriptase and therefore

affect the cDNA yield.

e) Short incubation time The standard reverse-transcription reaction

requires a 15-min incubation. In rare cases,

when analyzing RNAs with a very high degree

of secondary structure or if the RT-PCR product

is longer than 200 bp, it may be advantageous

to increase the incubation time to 30 min.

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QuantiTect Reverse Transcription Handbook 03/2009 15

Comments and suggestions

f) Poor quality or incorrect Check the concentration, integrity, and purity of

amount of template RNA for the template RNA (see Appendix B, page 20)

reverse-transcription reaction before starting the protocol. Mix well after

thawing the template RNA. Even minute amounts

of RNases can affect synthesis of cDNA and

sensitivity in RT-PCR, particularly with small

amounts of RNA.

g) RNA concentration too high Quantiscript Reverse Transcriptase is designed

or too low for use with 10 pg to 1 µg RNA. If using >1 µg

RNA, scale up the reaction linearly to the

appropriate volume.

h) RNA denatured Denaturation of the template RNA is not

necessary. If denaturation was performed, the

integrity of the RNA may be affected.

i) Incorrect concentration or If using a gene-specific primer for reverse

degradation of primers for transcription, check the concentration and

reverse-transcription reaction integrity of the primer. If necessary, perform

reverse transcription with different primer

concentrations or use the supplied RT Primer Mix.

If using RT Primer Mix, be sure to use 1 µl of RT

Primer Mix in a 20 µl reaction.

j) Incubation temperature Reverse transcription should be carried out at

too high 42°C. Higher temperatures may reduce the

length of cDNA products or the activity of

Quantiscript Reverse Transcriptase. Check the

temperature of your heating block or water bath.

FastLane Cell cDNA Kit users

k) Wrong protocol followed If using the QuantiTect Reverse Transcription Kit

to perform additional reverse-transcription

reactions with the FastLane Cell cDNA Kit,

follow the protocol in the

FastLane Cell cDNA

Handbook.

No product, or product detected late in real-time PCR, or only primer–dimers

detected (problems occurring during real-time PCR)

a) PCR annealing time too short Use the annealing time specified in the protocol

for the real-time PCR kit you are using.

b) PCR extension time too short Use the extension time specified in the protocol

for the real-time PCR kit you are using.

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QuantiTect Reverse Transcription Handbook 03/200916

Comments and suggestions

c) Mg

concentration in Always start with the Mg

concentration

PCR not optimal recommended in the protocol for the real-time

PCR kit you are using. Perform titration in

0.5 mM steps.

d) Pipetting error or missing Check the concentrations and storage conditions

reagent when setting up PCR of reagents, including primers and cDNA.

Taq

DNA Polymerase not Ensure that the cycling program includes the hot

activated with a hot start start activation step for

Taq

DNA polymerase; for

details, check the instructions supplied with the

polymerase.

f) PCR product too long For optimal results, PCR products should be

100–150 bp in length and should not exceed

300 bp.

g) Primer design for real-time Check for the presence of PCR products by gel

PCR not optimal electrophoresis or melting curve analysis. If no

specific PCR products are detected, review the

primer design.

h) Primer concentration Use the primer concentrations recommended in

for real-time PCR not optimal the protocol for the real-time PCR kit you are

using.

i) Insufficient number of cycles Increase the number of cycles.

j) PCR annealing temperature Decrease annealing temperature in 3°C steps.

too high

k) PCR annealing temperature Increase annealing temperature in 2°C steps.

too low

l) No detection activated Check that fluorescence detection was activated

in the cycling program.

m) Wrong detection step Ensure that fluorescence detection takes place

during the extension step of the PCR cycling

program.

n) Real-time PCR

Check for possible degradation of primers/probes

primers/probes degraded on a denaturing polyacrylamide gel.

o) Wrong dye layer/filter chosen

Ensure that the appropriate layer/filter is activated.

p) Insufficient starting template

Increase the amount of template cDNA, if possible.

q) Primer–dimers coamplified Include an additional data acquisition step in the

in real-time PCR with SYBR cycling program to avoid the detection of

Green I primer–dimers.

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QuantiTect Reverse Transcription Handbook 03/2009 17

Comments and suggestions

r) Detection temperature too Ensure that the detection temperature is at least

high in optional data 3°C lower than the

of the specific product.

acquisition step for real-time When establishing a new primer-template

PCR with SYBR Green I system, always perform a 3-step cycling reaction

first, without the optional fourth step.

Multiple peaks in melting temperature analysis/multiple PCR products

Reaction set up at room To avoid nonspecific primer annealing, set up the

temperature real-time PCR in cooled reaction vessels and/or

use a

Taq

DNA polymerase which requires a hot

start.

High fluorescence in “No RT” control reactions

Contamination with Check that the genomic DNA elimination step

genomic DNA with gDNA Wipeout Buffer was performed

correctly: check the temperature of your heating

block or water bath and the concentration of the

reaction components.

When purifying RNA, we recommend using

RNeasy

Plus Kits, which use gDNA Eliminator

columns or plates to remove genomic DNA

contamination (see page 29 for ordering

information).

No linearity in ratio of C

value/crossing point to log of the template amount

a) Template amount too high Do not exceed maximum recommended amounts

of template cDNA. For details, see the protocol

for the real-time PCR kit you are using.

b) Template amount too low Increase amount of template RNA, if possible.

High fluorescence in “No Template” control

a) Contamination of reagents Discard reaction components and repeat with

new reagents.

b) Contamination during Take appropriate safety precautions (e.g., use

reaction setup filter tips).

Varying fluorescence intensity

a) Real-time cycler contaminated Decontaminate the real-time cycler according to

the supplier’s instructions.

b) Real-time cycler no longer Recalibrate the real-time cycler according to the

calibrated supplier’s instructions.

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QuantiTect Reverse Transcription Handbook 03/200918

Appendix A: General Remarks on Handling RNA

Handling RNA

Ribonucleases (RNases) are very stable and active enzymes that generally do not

require cofactors to function. Since RNases are difficult to inactivate and even minute

amounts are sufficient to degrade RNA, do not use any plasticware or glassware

without first eliminating possible RNase contamination. Although the QuantiTect Reverse

Transcription Kit contains RNase inhibitor, we still recommend that care should be taken

to avoid inadvertently introducing RNases into the RNA sample during or after the

purification procedure. In order to create and maintain an RNase-free environment, the

following precautions must be taken during pretreatment and use of disposable and

nondisposable vessels and solutions while working with RNA.

General handling

Proper microbiological, aseptic technique should always be used when working with

RNA. Hands and dust particles may carry bacteria and molds and are the most common

sources of RNase contamination. Always wear latex or vinyl gloves while handling

reagents and RNA samples to prevent RNase contamination from the surface of the skin

or from dusty laboratory equipment. Change gloves frequently and keep tubes closed

whenever possible.

Disposable plasticware

The use of sterile, disposable polypropylene tubes is recommended throughout the

procedure. These tubes are generally RNase-free and do not require pretreatment to

inactivate RNases.

Nondisposable plasticware

Nondisposable plasticware should be treated before use to ensure that it is RNase-free.

Plasticware should be thoroughly rinsed with 0.1 M NaOH,* 1 mM EDTA* followed

by RNase-free water (see “Solutions”, page 19). Alternatively, chloroform-resistant

plasticware can be rinsed with chloroform* to inactivate RNases.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective

goggles. For more information, consult the appropriate material data sheets (MSDSs), available from the

product supplier.

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QuantiTect Reverse Transcription Handbook 03/2009 19

Glassware

Glassware should be treated before use to ensure that it is RNase-free. Glassware used

for RNA work should be cleaned with a detergent,* thoroughly rinsed, and oven baked

at 240°C for 4 hours or more (overnight, if more convenient) before use. Autoclaving

alone will not fully inactivate many RNases. Alternatively, glassware can be treated

with DEPC* (diethyl pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water),

allow to stand overnight (12 hours) at 37°C, and then autoclave or heat to 100°C for

15 minutes to eliminate residual DEPC.

Solutions

Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a

strong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of

0.1% to inactivate RNases on glass or plasticware or to create RNase-free solutions and

water. DEPC inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 ml

of the solution to be treated and shake vigorously to bring the DEPC into solution. Let

the solution incubate for 12 hours at 37°C. Autoclave for 15 minutes to remove any

trace of DEPC. DEPC will react with primary amines and cannot be used directly to treat

Tris* buffers. DEPC is highly unstable in the presence of Tris buffers and decomposes

rapidly into ethanol and CO

. When preparing Tris buffers, treat water with DEPC first,

and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC will

modify purine residues in RNA by carbethoxylation. Carbethoxylated RNA is translated

with very low efficiency in cell-free systems. However, its ability to form DNA:RNA or

RNA:RNA hybrids is not seriously affected unless a large fraction of the purine residues

have been modified. Residual DEPC must always be eliminated from solutions or vessels

by autoclaving or heating to 100°C for 15 minutes.

Note: QIAGEN solutions, such as Quantiscript RT Buffer and RNase-free water, are

guaranteed RNase-free without using DEPC treatment and are therefore free of any

DEPC contamination.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective

goggles. For more information, consult the appropriate material data sheets (MSDSs), available from the

product supplier.

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QuantiTect Reverse Transcription Handbook 03/200920

Appendix B: Preparation, Storage, Quantification,

and Determination of Quality of RNA

Preparation of RNA

Reverse transcriptases are used in vitro for first-strand cDNA synthesis with RNA as the

starting template. The efficiency of the reaction is highly dependent on the quality and

quantity of the starting RNA template.

It is important to have intact RNA as starting template. Even trace amounts of

contaminating RNases in the RNA sample can cause RNA cleavage, resulting in

shortened cDNA products. Chemical impurities, such as protein, poly-anions (e.g.,

heparin), salts, EDTA, ethanol, and phenol, can affect the activity and processivity of

the reverse transcriptase. To ensure reproducible and efficient reverse transcription, it is

important to determine the quality and quantity of the starting RNA (see below).

For best results, we recommend starting with RNA purified using silica-gel–membrane

technology. For ordering information, see page 29.

Storage of RNA

Purified RNA may be stored at –20°C or –70°C in water. Under these conditions, no

degradation of RNA is detectable after 1 year.

Quantification of RNA

The concentration of RNA should be determined by measuring the absorbance at

260 nm (

260

) in a spectrophotometer. To ensure significance, readings should be

greater than 0.15. An absorbance of 1 unit at 260 nm corresponds to 44 µg of RNA

per ml (

260

=1 → 44 µg/ml). This relation is valid only for measurements at a neutral

pH. Therefore, if it is necessary to dilute the RNA sample, this should be done in a buffer

with neutral pH. As discussed below (see “Purity of RNA”, page 21), the ratio between

the absorbance values at 260 and 280 nm gives an estimate of RNA purity.

When measuring RNA samples, be certain that cuvettes are RNase-free, especially if

the RNA is to be recovered after spectrophotometry. This can be accomplished by

washing cuvettes with 0.1 M NaOH,* 1 mM EDTA* followed by washing with

RNase-free water (see “Solutions”, page 19). Use the buffer in which the RNA is diluted

to zero the spectrophotometer.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective

goggles. For more information, consult the appropriate material data sheets (MSDSs), available from the

product supplier.

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QuantiTect Reverse Transcription Handbook 03/2009 21

An example of the calculation involved in RNA quantification is shown below:

Volume of RNA sample = 100 µl

Dilution = 20 µl of RNA sample + 180 µl of 10 mM Tris·Cl,* pH 7.0 (1/10 dilution)

Measure absorbance of diluted sample in a 0.2 ml cuvette (RNase-free):

260

= 0.2

Concentration of RNA sample = 44 µg/ml x

260

x dilution factor

= 44 µg/ml x 0.2 x 10

= 88 µg/ml

Total amount = concentration x volume of sample in ml

= 88 µg/ml x 0.1 ml

= 8.8 µg of RNA

Purity of RNA

The ratio of the readings at 260 nm and 280 nm (

260

280

) provides an estimate of

the purity of RNA with respect to contaminants that absorb in the UV, such as protein.

However, the

260

280

ratio is influenced considerably by pH. Since water is not

buffered, the pH and the resulting

260

280

ratio can vary greatly. Lower pH results in

a lower

260

280

ratio and reduced sensitivity to protein contamination.

†

For accurate

values, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure RNA

has an

260

280

ratio of 1.9–2.1

‡

in 10 mM Tris·Cl, pH 7.5. Always be sure to

calibrate the spectrophotometer with the same solution.

For determination of RNA concentration, however, we still recommend dilution of the

sample in a buffer with neutral pH since the relationship between absorbance and

concentration (

260

reading of 1 = 44 µg/ml RNA) is based on an extinction coefficient

calculated for RNA at neutral pH (see “Quantification of RNA”, page 20).

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective

goggles. For more information, consult the appropriate material data sheets (MSDSs), available from the

product supplier.

†

Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the

spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.

‡

Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some

spectrophotometers.

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QuantiTect Reverse Transcription Handbook 03/200922

Integrity of RNA

The integrity and size distribution of total RNA can be checked by denaturing agarose

gel electrophoresis and ethidium bromide staining* or by using the QIAxcel

system

(www

.qiagen.com/QIAxcel) or Agilent

2100 bioanalyzer. The respective ribosomal

RNAs should appear as sharp bands or peaks. The apparent ratio of 28S rRNA to 18S

rRNA should be approximately 2:1. If the ribosomal bands or peaks of a specific

sample are not sharp, but appear as a smear towards smaller sized RNAs, it is likely

that the sample suffered major degradation either before or during RNA purification.

The Agilent 2100 bioanalyzer also provides an RNA Integrity Number (RIN) as a useful

measure of RNA integrity. Ideally, the RIN should be close to 10, but in many cases

(particularly with tissue samples), RNA quality is greatly influenced by how well the

original sample was preserved.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective

goggles. For more information, consult the appropriate material data sheets (MSDSs), available from the

product supplier.

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QuantiTect Reverse Transcription Handbook 03/2009 23

Appendix C: Quantitative, Real-Time Two-Step RT-PCR

For the quantification of RNA transcripts, quantitative, real-time RT-PCR is the most

sensitive and reliable method. Real-time RT-PCR begins with the reverse transcription of

RNA into cDNA, and is followed by PCR amplification of the cDNA. RNA is transcribed

into single-stranded cDNA using random primers, gene-specific primers, or oligo-dT

primers that specifically hybridize to the poly-A tail of mRNAs. The quantity of cDNA is

determined during the exponential phase of PCR by the detection of fluorescence

signals that exceed a certain threshold. Fluorescence signals are generated by

fluorophores incorporated into the PCR product (e.g., in assays using SYBR Green I dye)

or by fluorophores which are coupled to short oligonucleotide probes (i.e., in probe-

based assays). In real-time RT-PCR, the level of RNA transcripts is calculated from the

number of the PCR cycle at which the threshold is exceeded. This cycle is called the

threshold cycle or the crossing point. For reliable results in quantitative, real-time PCR

of cDNA generated using the QuantiTect Reverse Transcription Kit, we recommend

using a Rotor-Gene Kit, QuantiFast Kit, or QuantiTect Kit (see page 10 for more

information).

In quantitative, real-time two-step RT-PCR, cDNA is first synthesized by reverse

transcription. An aliquot of the finished reverse-transcription reaction is then used for

PCR. Reverse transcription and PCR are performed sequentially in 2 separate reaction

tubes. With the QuantiTect Reverse Transcription Kit, RT Primer Mix (supplied) or gene-

specific primers (not supplied) can be used to synthesize cDNA for quantitative, real-

time two-step RT-PCR. In addition, cDNA can be stored for later analysis.

C1. Carry out reverse transcription according to the protocol on page 11, using the

QuantiTect Reverse Transcription Kit and 10 pg to 1 µg RNA.

C2. Add an aliquot of each finished reverse-transcription reaction to real-time PCR mix.

Note: No more than 1/10 of the final PCR volume should derive from the finished

reverse-transcription reaction. For example, for a 50 µl PCR assay, use ≤5 µl of the

finished reverse-transcription reaction.

C3. Carry out real-time PCR as recommended by the supplier.

We recommend using a Rotor-Gene Kit, QuantiFast Kit, or QuantiTect Kit (see

page 10).

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QuantiTect Reverse Transcription Handbook 03/200924

Appendix D: Recommended Controls for Quantitative,

Real-Time RT-PCR

No RT control

With the QuantiTect Reverse Transcription Kit, genomic DNA is efficiently removed in

a single step. However, all reverse-transcription experiments should include a negative

control to test for contaminating genomic DNA. Genomic DNA contamination can be

detected by performing a control reaction in which no reverse transcription is possible.

This control contains all components including template RNA, except for Quantiscript

Reverse Transcriptase. Reverse transcription therefore cannot take place and the only

template available is contaminating genomic DNA. In rare cases in which genomic

DNA is still amplified, detection of contaminating DNA can be eliminated with specially

designed primers or probes (Figure 2).

Primer spans an intron/exon boundary

Probe spans an intron/exon boundary

Figure 2. Primer/probe design. Primer/probe design to eliminate signals from contaminating genomic DNA.

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QuantiTect Reverse Transcription Handbook 03/2009 25

Positive control

In some cases, it may be necessary to include a positive control containing a known

concentration of template. This is usually a substitute for absolute standards and is

used only to test for presence or absence of the target, but does not yield detailed

quantitative information. Ensure that the positive control contains at least the minimum

amount of RNA required for accurate detection.

No template control (NTC)

All real-time PCR quantification experiments should include an NTC containing all the

components of the reaction except for the template. This enables detection of carryover

contamination from previous experiments.

References

QIAGEN maintains a large, up-to-date online database of scientific publications

utilizing QIAGEN products. Comprehensive search options allow you to find the articles

you need, either by a simple keyword search or by specifying the application, research

area, title, etc.

For a complete list of references, visit the QIAGEN Reference Database online at

www

.qiagen.com/RefDB/search.asp or contact QIAGEN Technical Services or your

local distributor.

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QuantiTect Reverse Transcription Handbook 03/200926

Ordering Information

Product Contents Cat. no.

QuantiTect Reverse For 10 x 20 µl reactions: gDNA 205310

Transcription Kit (10) Wipeout Buffer, Quantiscript Reverse

Transcriptase, Quantiscript RT Buffer,

RT Primer Mix, and RNase-Free Water

QuantiTect Reverse For 50 x 20 µl reactions: gDNA 205311

Transcription Kit (50) Wipeout Buffer, Quantiscript Reverse

Transcriptase, Quantiscript RT Buffer,

RT Primer Mix, and RNase-Free Water

QuantiTect Reverse For 200 x 20 µl reactions: gDNA 205313

Transcription Kit (200) Wipeout Buffer, Quantiscript Reverse

Transcriptase, Quantiscript RT Buffer,

RT Primer Mix, and RNase-Free Water

Accessories

QuantiTect Primer Assays — for use in real-time RT-PCR

with SYBR Green detection (search for and order assays at

www

.qiagen.com/GeneGlobe)

QuantiTect Primer For 200 x 50 µl reactions or Varies

Assay (200)* 400 x 25 µl reactions: 10x QuantiTect

Primer Assay (lyophilized)

Rotor-Gene SYBR Green PCR Kit — for ultrafast real-time PCR

and two-step RT-PCR using SYBR Green I on the Rotor-Gene Q

Rotor-Gene SYBR For 400 x 25 µl reactions: 3 x 1.7 ml 204074

Green PCR Kit (400)

†

2x Master Mix, 2 x 2 ml RNase-Free

Water

QuantiFast SYBR Green PCR Kit — for fast real-time PCR

and two-step RT-PCR using SYBR Green I

QuantiFast SYBR Green For 400 x 25 µl reactions: 3 x 1.7 ml 204054

PCR Kit (400)

†

2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free Water

* Assays also available in 96- or 384-well plates; please inquire.

†

Trial-size kit and larger kit also available; please inquire.

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QuantiTect Reverse Transcription Handbook 03/2009 27

Ordering Information

Product Contents Cat. no.

QuantiTect SYBR Green PCR Kit — for real-time PCR

and two-step RT-PCR using SYBR Green I

QuantiTect SYBR Green For 200 x 50 µl reactions: 3 x 1.7 ml 204143

PCR Kit (200)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free Water

Rotor-Gene Probe PCR Kit — for ultrafast real-time PCR

and two-step RT-PCR using sequence-specific probes

on the Rotor-Gene Q

Rotor-Gene Probe For 400 x 25 µl reactions: 3 x 1.7 ml 204374

PCR Kit (400)* 2x Master Mix, 2 x 2 ml RNase-Free

Water

QuantiFast Probe PCR Kits — for fast real-time PCR

and two-step RT-PCR using sequence-specific probes

For all instruments from Applied Biosystems except the Applied

Biosystems

7500

QuantiFast Probe PCR For 400 x 25 µl reactions: 3 x 1.7 ml 204254

Kit (400)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free Water

For the Applied Biosystems 7500 and instruments from other suppliers

QuantiFast Probe PCR For 400 x 25 µl reactions: 3 x 1.7 ml 204354

+ROX Vial Kit (400)* 2x Master Mix (without ROX dye),

210 µl ROX Dye Solution,

2 x 2 ml RNase-Free Water

QuantiTect Probe PCR Kit — for real-time PCR

and two-step RT-PCR using sequence-specific probes

QuantiTect Probe PCR For 200 x 50 µl reactions: 3 x 1.7 ml 204343

Kit (200)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free Water

Rotor-Gene Multiplex PCR Kit — for ultrafast multiplex

real-time PCR and two-step RT-PCR on the Rotor-Gene Q

Rotor-Gene Multiplex For 400 x 25 µl reactions: 3 x 1.7 ml 204774

PCR Kit (400)* 2x Master Mix, 2 x 2 ml RNase-Free

Water

* Trial-size kit and larger kit also available; please inquire.

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QuantiTect Reverse Transcription Handbook 03/200928

Ordering Information

Product Contents Cat. no.

QuantiFast Multiplex PCR Kits — for fast multiplex real-time

PCR and two-step RT-PCR

For all instruments from Applied Biosystems except the Applied

Biosystems 7500

QuantiFast Multiplex For 400 x 25 µl reactions: 3 x 1.7 ml 204654

PCR Kit (400)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free Water

For the Applied Biosystems 7500 and instruments from other suppliers

QuantiFast Multiplex For 400 x 25 µl reactions: 3 x 1.7 ml 204754

PCR +R Kit (400)* 2x Master Mix (without ROX dye),

210 µl ROX Dye Solution,

2 x 2 ml RNase-Free Water

QuantiTect Multiplex PCR Kits — for multiplex real-time PCR

and two-step RT-PCR

For all instruments from Applied Biosystems

QuantiTect Multiplex For 200 x 50 µl reactions: 3 x 1.7 ml 204543

PCR Kit (200)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free Water

For instruments from other suppliers

QuantiTect Multiplex For 200 x 50 µl reactions: 3 x 1.7 ml 204743

PCR NoROX Kit (200)* 2x Master Mix (without ROX dye),

2 x 2 ml RNase-Free Water