Quantitect Reverse Transcription Kit For cDNA Synthesis From Qiagen

Qiagen’s Quantitect Reverse Transcription Kit is designed to generate high quality cDNA, which can be used for highly sensitive downstream applications such as real-time quantitative PCR (qPCR). Qiagen markets this kit specifically for qPCR which makes it very appealing to researchers doing real-time gene expression studies. The way Qiagen has achieved this appeal is to integrate a genomic DNA (gDNA) removal step just prior to cDNA synthesis. This is important because gDNA contamination in a cDNA sample will not reflect the transcribed mRNA but instead, just the presence or absence of a gene. In order to do qPCR and accurately study gene expression, it is critical to generate starting material (cDNA) that is not contaminated with gDNA.

The kit itself is comprised of a series of steps that do not take very long, and this is beneficial while working with RNA. The reason for this, of course, is that RNA degrades faster than other biological macromolecules because of highly stable and fast RNase activity. RNA samples are very sensitive to degradation if kept at temperatures where there is even minimal activity of RNase enzymes, which are present abundantly in general lab dust. I myself use this kit routinely to generate cDNA from RNA samples that have been generated from frozen Arabidopsis leaf tissue. I work with several different mutants and study the expression of genes in different genetic backgrounds. The Quantitect kit has a very fast gDNA removal step which takes only a couple of minutes followed immediately by a reverse transcription step, which means that RNA is converted to cDNA quickly, consequently limiting the possibility of degradation.

The kit has been designed to generate cDNA from 1 ug of RNA starting material and this can be scaled up. One interesting addition is the presence of RNase H in the reverse transcription mix. RNase H is different from general RNase enzymes in that it only degrades RNA that is bound to DNA, so it removes RNA from RNA:DNA hybrids which are generated during cDNA synthesis, but doesn’t act on free RNA which is used as template for cDNA. The kit also comes with RNase inhibitors and dNTP’s as well as a universal primer mix to enable cDNA strand synthesis. The entire experiment can be done in the space of an hour, and possibly faster if RNA samples do not need any pre-dilution or mixing. I would certainly recommend the use of this kit as I haven’t had any problems with it and the quality of cDNA has generally proved to be high and consistent in amounts and performance.