Transcriptor First Strand cDNA Synthesis Kit From Roche

Reverse transcription PCR (RT-PCR) is now a crucial application in many labs, allowing for highly sensitive amplification of specific RNA sequences. Reverse transcription kits make this fundamental process fast and effective, supplying the need for sensitive, cost effective and reliable methods.

The Transcritpor first strand cDNA synthesis kit by Roche has been specifically designed to reverse transcribe a variety of RNA species (mRNA, total RNA, viral RNA and in vitro-transcribed RNA) from a variety of sources, for further use in real-time PCR, generating cDNA libraries and cloning genes of interest. The kit is capable of reverse transcribing notoriously difficult templates (up to 70% GC-rich/secondary structures) with high reproducibility. It is possible to generate cDNA transcripts up to 14 kb using either random hexamer primers, anchored oligo(dT)₁₈ primers (both included in the kit) or user-supplied sequence specific primers.

Everything required to carry out the reverse transcription is included in the kit (Transcriptor reverse transcriptase, Transcriptor RT reaction buffer, Protector RNase Inhibitor, Deoxynucleotide mix, anchored oligo(dT) ₁₈ primer and random hexamer primer). Also included is a control RNA and control PBGD primer mix. This allows the user to validate the kit and methodology before moving on to precious RNA samples. Two alternative protocols are supplied with the kit, allowing the user to use either anchored oligo(dT) ₁₈, random hexamer or sequence specific primers independently, or alternatively a combination of anchored oligo(dT) ₁₈ and random hexamer primers, allowing for a reaction with increased sensitivity. The reaction is sensitive enough to reverse transcribe 10 ng to 5 ug of total RNA or 1 to 100 ng of mRNA starting material.

I have used both random hexamer and sequence specific primers to amplify from 100 ng of total RNA. In both cases, the an initial 13 µl volume master mix was prepared in a thin-walled PCR tube using 2 ul total RNA, 2 ul random hexamer or sequence specific primers and 9 ul PCR grade water, all of which were briefly centrifuged prior to use. The template-primer mixture reaction tubes were then heated at 65°C for 10 minutes in a thermal block cycler with heated lid, in order to denature RNA secondary structures. The tubes were then immediately cooled on ice. To this reaction mixture, the following reverse transcription components were added: Transcriptor Reverse Transcriptase Reaction buffer (4 ul), Protector RNase Inhibitor (0.5 ul), deoxynucleotide mix (2 ul) and Transcriptor Reverse Transcriptase (0.5 ul), to bring the final volume up to 20 ul. The reaction components were carefully mixed by inverting and centrifuged briefly to collect the sample. The tubes were then placed in a thermal block cycler with heated lid and subjected to the program of heating outlined in the protocol. The reaction was stopped by placing reaction tubes onto ice and whole process took between 70 – 90 minutes, depending on the number of samples to be processed.

I have used the kit a number of times over the past 1.5 years and found the results to be consistent and reliable. I use the cDNA for expression analysis in quantitative real-time PCR and to date, have never had a problem with the cDNA produced using this kit. The only real disadvantage is that the cDNA product is limited to 20 ul. The turnaround time however is rapid and the protocols included in the kit are easy to follow. In addition, all reaction components required are included in the kit, which can be bought in both 100 and 200 reaction sizes.