The iScript cDNA Synthesis Kit from Bio-Rad provides a simple means of producing cDNA for cloning purposes or real-time PCR. The kit consists of three tubes: buffer, iScript reverse transcriptase and nuclease-free water. The iScript reverse transcriptase is an RNaseH+ modified, MMLV-derived enzyme mixed with an RNase inhibitor. The H+ aspect of the RNase allows greater sensitivity in detecting RNA transcripts and, therefore, less RNA (as little as 100 fg according to manufacturer). The iScript kit uses a blend of oligo (dT) and random hexamers to prime cDNA synthesis. To generate cDNA, the components (along with the sample RNA) are subjected to three incubations: 25C for 5 minutes, 42C for 30 minutes and 85C for 5 minutes. A PCR machine is easily programmed to perform these incubations. The reactions are then ready for downstream applications.
I have been using this kit for real-time PCR with great satisfaction since it was first introduced. Previously, I had used the Superscript II Kit from Invitrogen with several more components and multiple incubation steps that required a water bath dance. I compared the performance of the two kits in generating cDNA for real-time PCR. Specifically, I isolated RNA from T helper cells, generated cDNA from 1 ug of RNA and then performed real-time PCR to determine the presence of cytokine transcripts using dual-labeled fluorescent probes. I found the two kits performed equally well for this application, detecting the same quantities of transcripts from the cDNA samples. In contrast, the iScript kit was less expensive, faster (iScript - 40 minutes, Superscript - 83 minutes) and far less labor intensive with the use of PCR machine programmed incubations than the Invitrogen competitor. Furthermore, I have only used this kit for the real-time PCR application, but Bio-Rad claims that the iScript is a high fidelity enzyme capable of generating cDNA’s greater than 6Kb. However, since the iScript mastermix contains both random hexamer and oligo(dT) to prime cDNA synthesis, there may be a lower yield of full-length cDNAs corresponding to polyA containing mRNAs.
Kerri Mowen, Ph.D.
Assistant Professor
Department of Immunology
The Scripps Research Institute