cDNA synthesis using the QuantiTect Reverse transcription kit

Enrico Bagnoli, Miratul Muqit

Published: 2024-05-14 DOI: 10.17504/protocols.io.q26g718mkgwz/v1

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Abstract

This protocol details the synthesis of cDNA.

Steps

cDNA synthesis

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°C``25°C).

Mix each solution by flicking the tubes. Centrifuge briefly to collect residual liquid from the sides of the tubes, and then keepOn ice .

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.The protocol is for use with 10 pg to 1µg RNA. If using >1 μg RNA, scale up the reaction linearly. For example, if using 2µg RNA, double the volumes of all reaction components for a final 28µL reaction volume.

Incubate for 0h 2m 0s at 42°C, then place immediately On ice.

Note

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

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.If using >1 μg RNA, scale up the reaction linearly. For example, if using 2µg RNA, double the volumes of all reaction components for a final 40µL reaction volume.

Add template RNA from step 3 (14µL) to each tube containing reverse-transcription master mix. Mix and then store On ice.

Incubate for 0h 15m 0s 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 0h 30m 0s may increase cDNA yields.

Incubate for 0h 3m 0sat 95°C to inactivate Quantiscript Reverse Transcriptase.

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.

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cDNA synthesis using the QuantiTect Reverse transcription kit

Abstract

Steps

cDNA synthesis

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