ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis From Invitrogen

Reverse Transcriptase (RT)-PCR of RNA with extensive secondary structures, or the detection of rare messages can be challenging and laborious. Another critical point is the RNase H activity of the reverse transcriptase used in the respective assay. Therefore, an RT system is sometimes needed which has low RNase activity and is able to effectively produce first-strand cDNA from highly structured as well as rare samples.

The ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis from Invitrogen includes ThermoScript™ RT, which is an avian reverse transcriptase with reduced RNase H activity and which is engineered to have higher thermal stability. The enzyme will produce higher yields of cDNA, and produce more full-length cDNA transcripts than regular AMV RT. The RT can be upgraded by combination with high performance Platinum® Taq DNA polymerase. The system is then suitable for the detection of RNA targets from 100 bp to greater than 12kb.

I use this RT kit in combination with the Platinum® Taq DNA polymerase for the detection of rather rare messages. For some in vitro transcription experiments, I generate the template by a PCR approach. In these cases, I also use the ThermoScript™ RT system for accurate template production.

The RT kit contains all the necessary reagents to run 25 amplification reactions. The user has the choice of using total RNA or poly(A)-selected RNA as a template. ThermoScript™ RT works with oligo(dT), random primers or a gene specific primer, which are included in the kit except for the gene specific primers which have to be provided by the user. In addition, the kit contains dNTPs, RNaseOUT™, an RNase inhibitor, and RNase H to remove the RNA template after the RT reaction.

For the cDNA synthesis reaction, template RNA, dNTPs and primer (oligo(dT), random hexamers, or gene specific primer) are combined and adjusted to 12 ul with nuclease-free water. After 5 minutes of incubation at 65ºC, the samples are set on ice. One important detail is to vortex the 5x cDNA synthesis buffer for 5 seconds just prior use; since the buffer is viscous, this should not be forgotten. Next, the master mix containing the synthesis buffer, DTT, RNase inhibitor, nuclease-free water and the ThermoScript™ RT enzyme are added to the sample and incubated following protocols depending on the primer species used. In addition, the protocol recommends the adjustment of the amount of RT enzyme added to the reaction depending on the amount of RNA which will be reverse transcribed. At the end of the procedure, there is an optional step of RNase treatment to eliminate the RNA template. The manufacturers protocol also includes further processing notes using Platinum® Taq DNA polymerase for downstream PCR amplification.

In summary, the kit works really well for hard to amplify or rare messages, is straightforward to use, especially if combined with the matching PCR enzymes.