Bio-Rad’s iScript cDNA Synthesis Kit provides everything required for reverse transcription; 5x transcription mix, nuclease-free water and iScript reverse transcriptase. It is designed to enable two-step RT-PCR analysis with high sensitivity over a wide range of RNA sources and optimized for amplification of targets less than 1 kb in size. The iScript enzyme is a modified MMLV-reverse transcriptase with RNase H+ activity. RNase inhibitor, a blend of oligo(dT) and random hexamer primers and dNTPs are all included in the 5x transcription mix, which minimizes both pipetting and time requirements for setup. The kit allows researchers to run extremely convenient and consistent reverse transcription reactions, as everything is assembled in the tubes at once without requiring separate steps for heat denaturation, chilling or primer annealing (minimizing the chance of cross-contamination). Once the reactions are mixed and spun, the tubes are incubated for 5 min at 25C, for 30 min at 42C and for 5 min at 85C. The competitively priced kit is provided in two sizes (25 or 100 reactions). The documentation is succinct, but sufficient.
I have been using the iScript cDNA Synthesis Kit primarily for quantitative real-time RT-PCR analyses. I found it to be well-suited for standard PCR with post-amplification analysis in agarose gels with ethidium bromide staining, as well. I used this kit to make cDNA from total RNA isolated from established cell lines (human lung epithelial cell line A549 and mouse lymphoma cell line EL4), mouse primary T lymphocytes and tissues (lungs and livers). The simplicity of the protocol allows me to run multiple reactions for first-strand cDNA synthesis in PCR tubes or multi-well plates. If more than 5 reactions are required, I usually make the master mix (iScript enzyme plus 5x transcription mix) and add it to the wells (5 ul/reaction), which are pre-filled with water. Following RNA addition, the plates are sealed and cDNA is synthesized in a thermal cycler without any additional attention requirement. The kit has shown great sensitivity for low abundance and complex targets, even when the initial RNA amount is significantly less than 1 ug. The only disadvantage of the iScript cDNA Synthesis Kit is that it should be used with caution if researchers test the effects of RNA interference (siRNA). The presence of random hexamer primers in the reaction may allow cDNA synthesis and subsequent PCR amplification from partially degraded or decayed RNA. Perhaps, Bio-Rad’s iScript Select cDNA Synthesis kit is a better choice for such applications.
Timur Yarovinsky, PhD
Associate Research Scientist
The University of Iowa