AccuScript High Fidelity 1st Strand cDNA Synthesis Kit

AccuScript High Fidelity 1

Strand

cDNA Synthesis Kit

INSTRUCTION MANUAL

Catalog #200820

Revision C.0

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

200820-12

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AccuScript High Fidelity 1

Strand cDNA Synthesis Kit

CONTENTS

Materials Provided .............................................................................................................................. 1

Storage Conditions .............................................................................................................................. 1

Additional Materials Required .......................................................................................................... 1

Introduction ......................................................................................................................................... 2

Preprotocol Considerations ................................................................................................................ 2

RNA Isolation ........................................................................................................................ 2

cDNA Synthesis Primers ....................................................................................................... 3

cDNA Synthesis Reaction ..................................................................................................... 3

The Reverse Transcriptase-Mediated Polymerase Chain Reaction Protocol ................................ 4

Synthesis of First-Strand cDNA Using Reverse Transcriptase ............................................. 4

Amplification of First-Strand cDNA using the Polymerase Chain Reaction ........................ 6

Troubleshooting .................................................................................................................................. 8

References ............................................................................................................................................ 8

MSDS Information .............................................................................................................................. 8

Quick-Reference Protocol ................................................................................................................ 10

AccuScript High Fidelity 1

Strand cDNA Synthesis Kit

ATERIALS PROVIDED

Materials provided Concentration Quantity

AccuScript High Fidelity RT — 50 reactions

AccuScript RT buffer

10× 100 µl

Oligo(dT) primer (18-mers) 0.5 µg/µl 25 µg

Random primers (9-mers) 0.1 µg/µl 15 µg

100 mM dNTP Mix 25 mM each dNTP 40 µl

100 mM DTT 100 mM 100 µl

RNase-free water — 1.2 ml

RNase Block 40 U/µl 1000 U

The AccuScript high fidelity 1

strand cDNA synthesis kit provides enough reagents for 50 reactions.

The 10× AccuScript RT buffer contains 0.5 M Tris-HCl (pH 8.3), 0.75 M KCl, 0.03 M MgCl

STORAGE CONDITIONS

All Reagents: –20°C

ADDITIONAL MATERIALS REQUIRED

PfuUltra DNA polymerase & reaction buffer (optional)

Thin-walled PCR tubes (optional)

Revision C.0 © Agilent Technologies, Inc. 2015.

AccuScript High Fidelity 1

Strand cDNA Synthesis Kit 1

INTRODUCTION

Reverse transcriptases exhibit significantly higher error rates than other

known DNA polymerases, introducing errors at frequencies of one per 1,500

to 30,000 nucleotides during cDNA synthesis.

To solve this problem, we

developed AccuScript reverse transcriptase, a Moloney murine leukemia

virus reverse transcriptase (MMLV-RT) derivative combined with a

proofreading 3’-5’ exonuclease.

AccuScript reverse transcriptase delivers

the highest reverse-transcription accuracy while promoting full length

cDNA synthesis and superior performance in RT-PCR. AccuScript reverse

transcriptase delivers greater than three-fold higher accuracy compared to

leading reverse transcriptases, representing a significant advancement in

cDNA synthesis accuracy. Additionally, the AccuScript high fidelity 1

strand cDNA synthesis kit applies this superior performance to the

amplification of specific complementary DNA (cDNA) fragments from

limited amounts of RNA using a reverse transcriptase-mediated polymerase

chain reaction (RT-PCR) which results in high yield, full length cDNA. The

first-strand synthesis protocol generates a heterogeneous population of

cDNA molecules from all available poly(A)+ mRNA or total RNA, while

subsequent amplification with sequence-specific primers yields a

homogeneous population of the specific cDNA of interest, eliminating the

need for amplification and screening of a cDNA library. Resulting

populations of cDNA can be used for microarray, conventional and real time

PCR amplification.

The AccuScript high fidelity 1

strand cDNA synthesis kit delivers robust

first strand cDNA, superior RT-PCR yields from low RNA input amounts

and exceptional full-length cDNA capability—making it the perfect choice

for any application requiring premium first strand cDNA.

PREPROTOCOL CONSIDERATIONS

RNA Isolation

High-quality intact RNA is essential for successful synthesis of full-length

cDNA and yield of long RT-PCR products. Total and poly(A)

RNA can be

rapidly isolated and purified using the Stratagene Absolutely RNA

purification kits. Oligo(dT)-selection for poly(A)

RNA is typically not

necessary, although including this step may improve the yield of specific

cDNA templates. RNA samples with an OD

260/280

of 1.8–2.0 are optimal.

Take precautions to minimize the potential for contamination by

ribonucleases (RNases). RNA isolation should be performed under

RNase-free conditions. Wear gloves and use sterile tubes, pipet tips, and

RNase-free or DEPC-treated water. (Although DEPC-treated water may be

used for RNA isolation, use the RNase-free water provided, instead of

DEPC-treated water, as the water component in the cDNA synthesis

reaction since DEPC can inhibit PCR.) Use of an RNase inhibitor, such as

Stratagene RNase Block Ribonuclease Inhibitor, is recommended when

isolating RNA from samples high in RNase activity.

2 AccuScript High Fidelity 1

Strand cDNA Synthesis Kit

cDNA Synthesis Primers

Oligo(dT)

is recommended for priming polyadenylated RNA and is

provided with this kit. Use of Oligo(dT)

allows the subsequent

amplification of products of multiple transcripts from a single first-strand

synthesis reaction. Random 9-mers, also provided with this kit, are efficient

primers for the detection of multiple short RT-PCR targets. If random

9-mers are used, the first-strand synthesis reaction must be incubated at

25°C for 10 minutes to extend the primers prior to increasing the reaction

temperature to 42°C for cDNA synthesis. Gene-specific primers anneal only

to defined sequences and are used to synthesize cDNA from particular

mRNA transcripts rather than from the entire mRNA population in the

sample. Specificity of priming with gene-specific primers may be improved

by optimizing annealing and reaction temperatures.

cDNA Synthesis Reaction

Incubation Temperature and Duration

Denaturation of the RNA template and primer by incubating the reaction at

65°C for 5 minutes is essential.

AccuScript RT is effective between 37 and 42°C. A 60-minute incubation

for the first-strand synthesis reaction is recommended for most targets. A

shorter incubation time (15–30 minutes) may be sufficient for some targets

and applications. Rare RNA sequences, long transcripts, or targets at the 5´

end of long transcripts benefit from a longer incubation at 42°C (up to

90 minutes).

AccuScript RT Inhibition of PCR

AccuScript RT can inhibit subsequent PCR and is inactivated by incubation

at 70°C after the cDNA synthesis reaction is complete. For long RNA

targets, it is advisable to increase the incubation time for reverse

transcription rather than increasing the amount of AccuScript RT in

the reaction.

RNase Inhibitor

We recommend adding RNase Block RNase inhibitor (20 U per 20-µl

reaction) to the first-strand synthesis reaction, as specified in the following

protocol. The use of RNase inhibitor at higher concentrations may reduce

product yield. If RNase inhibitor is omitted from the reaction, increase the

volume of water accordingly.

AccuScript High Fidelity 1

Strand cDNA Synthesis Kit 3

THE REVERSE TRANSCRIPTASE-MEDIATED POLYMERASE CHAIN

REACTION PROTOCOL

Note Wear gloves at all times during the first-strand cDNA synthesis

and PCR amplification procedures and while handling materials

and equipment to prevent contamination by ribonucleases

(RNases).

Synthesis of First-Strand cDNA Using Reverse Transcriptase

Note Mix and spin each component in a microcentrifuge before use.

1. Prepare the cDNA synthesis reaction by adding the following

components to a microcentrifuge tube in order:

RNase-free water to total volume 16.5 µl

2.0 µl of AccuScript RT Buffer (10×)

1.0 µl of a gene-specific primer (0.1 µg /µl) OR oligo(dT) primer

(0.5 µg/µl) OR 3 µl of random primers (0.1 µg/µl)

0.8 µl of dNTP mix (25 mM each dNTP)

X µl of RNA. The quantity of RNA depends on the RNA purity,

message abundance, and size of the target:

RNA Quantity

Total RNA, target <2 kb 10–200 ng

Total RNA, target >2 kb 200–5000 ng

mRNA (all targets) 0.1–100 ng

2. Incubate the reaction at 65°C for 5 minutes.

3. Cool the reaction at room temperature to allow the primers to anneal to

the RNA (approximately 5 minutes).

4. Add the following components to the reaction, in order, for a final

reaction volume of 20 µl:

2 µl of 100 mM DTT

1 µl of AccuScript RT

0.5 µl of RNase Block ribonuclease inhibitor (40 U/ µl)

Note To prevent heat inactivation, AccuScript RT and RNase Block

must be added after the reactions have cooled to room

temperature following the 65°C incubation.

5. If using random primers, incubate the reaction at 25°C for 10 minutes to

extend the primers prior to the 42°C synthesis step. If using oligo(dT) or

gene-specific primers, proceed to step 6.

4 AccuScript High Fidelity 1

Strand cDNA Synthesis Kit

6. Place the tube in a temperature-controlled thermal block at 42°C and

incubate the reaction for 60 minutes.

7. Terminate cDNA synthesis by incubating the reaction at 70ºC for

15 minutes.

8. Place the completed first-strand cDNA synthesis reaction on ice for use

in downstream applications. If performing RT-PCR, proceed to the

PCR amplification protocol (see Amplification of First-Strand cDNA

Using the Polymerase Chain Reaction). For long-term storage, place

the reaction at –20°C.

AccuScript High Fidelity 1

Strand cDNA Synthesis Kit 5

Amplification of First-Strand cDNA using the Polymerase Chain

Reaction

Notes cDNA synthesis reaction products should be stored at –20°C until

needed.

Mix and spin each component in a microcentrifuge before use.

A protocol for amplification using Stratagene high-fidelity

PfuUltra DNA polymerase is detailed below. This protocol could

be adapted to other PCR enzymes. Amplification with a high-

fidelity PCR enzyme is strongly recommended, however, in order

to realize the benefits of high-fidelity reverse transcription

provided by AccuScript RT.

Amplification using PfuUltra DNA Polymerase

1. Add the following components in order to sterile thin-walled PCR

tubes

for each PCR amplification reaction:

37.6 µl of RNase-free water

5 µl of 10× PfuUltra PCR buffer

2 µl of 25 mM MgSO

0.4 µl of dNTP mix (25 mM each dNTP)

1 µl of upstream primer (0.1 µg/µl)

1 µl of downstream primer (0.1 µg/µl)

2 µl of experimental first-strand cDNA reaction

1 µl of PfuUltra DNA polymerase (2.5 U/µl)

2. If the thermal cycler does not have a hot top assembly, overlay each

reaction with one or two drops (20–40 µl) of nuclease-free mineral oil

to prevent evaporation and condensation during thermal cycling.

The PfuUltra reaction buffer (supplied with the enzyme) contains MgSO

at a final

concentration of 2 mM. For amplification of cDNA using this enzyme, the reaction mixture

should be supplemented with MgSO

to a final concentration of 3 mM.

Thin-walled tubes are highly recommended for use with Stratagene thermal cyclers. These

tubes ensure ideal contact with the multiblock design, permit efficient heat transfer, and

maximize thermal-cycling performance.

6 AccuScript High Fidelity 1

Strand cDNA Synthesis Kit

3. Place the amplification reactions in a thermal cycler, and run the

thermal-cycling program below.

PCR Cycling Program for PfuUltra DNA Polymerase in RT-PCR

Thermal cycler Cycles Temperature Duration

Single or multiple block 1 95°C 1 minute

40 95°C 30 seconds

–5°C

30 seconds

68°C 3 minutes/kb

1 68°C 10 minutes

RoboCycler temperature

cycler

1 95°C 1 minute

40 95°C 1 minute

–5°C

1 minute

68°C 3 minutes/kb

1 68°C 10 minutes

Use the annealing temperature appropriate for the specific primer pair used in the

reaction.

For targets <1 kb, use a 3-minute extension time.

4. Analyze the PCR products by loading 10 µl of each PCR amplification

reaction (taken from below the mineral oil layer) into separate lanes of

a 0.8% (w/v) agarose gel.

AccuScript High Fidelity 1

Strand cDNA Synthesis Kit 7

TROUBLESHOOTING

Observation Suggestion

No or low yield of first-strand cDNA Verify the integrity of the RNA by denaturing agarose gel electrophoresis to

ensure it is not degraded.

Replace the RNA. Use Stratagene Absolutely RNA or Absolutely mRNA

purification kits to isolate intact total RNA or mRNA, respectively.

Isolate the RNA in the presence of a ribonuclease inhibitor, and ensure that all

RT-PCR reagents and labware are free of RNases.

Reduce the volume of the target RNA or remove RT inhibitors (SDS, EDTA,

guanidinium chloride, formamide, Na

, or spermidine) with an additional

70% (v/v) ethanol wash following ethanol precipitation.

In some cases RNase Block ribonuclease inhibitor can inhibit the cDNA

reaction. Reduce or eliminate the RNase Block.

Increase the length of the 42°C cDNA synthesis reaction to 90 minutes to

allow for the synthesis of cDNA from rare or long RNA targets.

Increase the concentration of the template RNA.

Add the AccuScript RT after the reactions have cooled to room-temperature

following the 65°C denaturation step, and synthesize cDNA at 42°C.

Confirm that the cDNA synthesis primer is complementary to the target

sequence; change the primer type [oligo(dT), gene-specific, or random].

If using random primers, incubate the reaction at 25°C for 10 minutes prior to

increasing the temperature to 42°C for cDNA synthesis. This allows better

annealing of random primers to RNA.

In the case of eukaryotic RNA, use the oligo(dT) primer.

REFERENCES

1. Roberts, J. D., Bebenek, K. and Kunkel, T. A. (1988) Science 242:1171-1173.

2. Arezi, B. and Hogrefe, H. H. (2007) Anal Biochem 360(1):84-91.

MSDS INFORMATION

The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on the web at

http://www.stratagene.com/MSDS/. Simply enter the catalog number to retrieve any associated MSDS’s

in a print-ready format. MSDS documents are not included with product shipments.

8 AccuScript High Fidelity 1

Strand cDNA Synthesis Kit

AccuScript High Fidelity 1

Strand cDNA Synthesis Kit

Catalog #200820

QUICK-REFERENCE PROTOCOL

cDNA Synthesis

♦ Add the following reagents, in order, to a microcentrifuge tube:

Reagent Volume ( for 20 µl final reaction volume)

RNase-free water To final volume of 16.5 µl

10 × AccuScript RT buffer 2.0 µl

Primer 1 µl gene-specific primer (0.1 µg/µl)

OR 1 µl oligo(dT) primer (0.5 µg/µl)

OR 3 µl random primers (0.1 µg/µl)

dNTP mix (25 mM each) 0.8 µl

Sample RNA X µl

♦ Incubate the reaction at 65°C for 5 minutes, then cool to room temperature (approximately

5 minutes).

♦ Add the following components, in order:

2 µl of 100 mM DTT

1 µl of AccuScript RT

0.5 µl of RNase Block

♦ If using random primers, incubate the reactions at 25°C for 10 minutes to allow primer

extension prior to completing the following step.

♦ Incubate the reaction at 42°C for 60 minutes.

♦ Terminate cDNA synthesis by incubating the reaction at 70ºC for 15 minutes. Place the

reaction on ice for use in downstream applications or at –20°C for long-term storage.