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Protocol CDNA

The QuantiTect Reverse Transcription Kit should be stored at –30 to –15°C and includes components for reverse transcription reactions. The protocol outlines steps for genomic DNA elimination, preparation of reverse-transcription master mix, and incubation conditions to ensure high cDNA yields. For further details, users are directed to the QuantiTect Reverse Transcription Handbook and other resources provided by QIAGEN.

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20 views4 pages

Protocol CDNA

The QuantiTect Reverse Transcription Kit should be stored at –30 to –15°C and includes components for reverse transcription reactions. The protocol outlines steps for genomic DNA elimination, preparation of reverse-transcription master mix, and incubation conditions to ensure high cDNA yields. For further details, users are directed to the QuantiTect Reverse Transcription Handbook and other resources provided by QIAGEN.

Uploaded by

anyaseradj
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Quick-Start Protocol March 2016

QuantiTect® Reverse Transcription Kit


The QuantiTect Reverse Transcription Kit (cat. nos. 205310, 205311, 205313 and 205314)
should be stored immediately upon receipt at –30 to –15°C in a constant-temperature freezer.

Further information

 QuantiTect Reverse Transcription Handbook: www.qiagen.com/HB-0189


 Safety Data Sheets: www.qiagen.com/safety
 Technical assistance: support.qiagen.com

Notes before starting

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


incubate at 37°C until the precipitates dissolve.
 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 or 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.
For gene-specific primers, we recommend using a final concentration of 0.7 µM.

 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 min.

Sample to Insight__
 If working with RNA for the first time, refer to Appendix A of the QuantiTect Reverse
Transcription Handbook.

 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 this protocol.

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 keep on ice.

2. Prepare the genomic DNA elimination reaction on ice according to Table 1. Mix and
then keep on ice.
Note: If setting up more than one reaction, prepare a master mix of gDNA Wipeout
Buffer and RNase-free water with a volume 10% greater than that required for the total
number of reactions to be performed. Distribute the appropriate volume of master mix
into individual tubes, followed by each RNA sample.

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

gDNA Wipeout Buffer, 7x 2 µl

Template RNA, up to 1 µg* Variable

RNase-free water Variable

Total reaction 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
keep 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. Distribute the
appropriate volume into individual tubes.
Note: 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.
Table 2. Reverse-transcription reaction components

Component Volume/reaction

Reverse-transcription master mix


Quantiscript Reverse Transcriptase* 1 µl

Quantiscript RT Buffer, 5x †‡
4 µl

RT Primer Mix ‡
1 µl

Template RNA
Entire genomic DNA elimination reaction (step 3) 14 µl (added at step 5)

Total reaction volume 20 µl

* Also contains RNase inhibitor.


†
Includes Mg2+ 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.
Note: 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. Place the reverse-transcription reactions on ice and proceed directly with real-time PCR.
For long-term storage, store reverse-transcription reactions at –20°C.
Note: For details on performing real-time PCR after reverse transcription, refer to
Appendix C of the QuantiTect Reverse Transcription Handbook. For details on
appropriate controls, see Appendix D. We recommend using a Rotor-Gene® Kit,
QuantiFast® Kit or QuantiTect Kit for real-time PCR.

Scan QR code for handbook.

For up-to-date licensing information and product-specific disclaimers, see the respective
QIAGEN kit handbook or user manual.

Trademarks: QIAGEN®, Sample to Insight®, FastLane®, QuantiFast®, Quantiscript®, QuantiTect®, Rotor-Gene® (QIAGEN Group). 1101351 03/2016 HB-0770-002
© 2016 QIAGEN, all rights reserved.

Ordering www.qiagen.com/contact | Technical Support support.qiagen.com | Website www.qiagen.com

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