iScript Reverse Transcription Supermix for RT-qPCR from Bio-Rad Laboratories

Bio-Rad’s iScript™ Reverse Transcription Supermix is sensitive, fast, and convenient for gene expression analysis using real-time RT-qPCR. It makes it really easy to convert mRNA into cDNA for the RT-PCR.  This kit is a pre-mixed 5x supermix; it contains all the necessary components, including reaction buffer, iScript MMLV reverse transcriptase, RNase inhibitor, dNTPs, oligo(dT) primer, random primers, MgCl2, and stabilizers for reverse transcription in one single tube.  It has everything you need except the RNA!

I tested Bio-Rad’s iScript™ Reverse Transcription Supermix for the preparation of transcripts for subsequent quantification of gene expression in embryonic stem cells (to show the pluripotent gene expression level) and cancer cell lines (to show the inflammantory gene expression level). It worked very well among the different cell lines I tested: human embryonic stem cells, human fibroblast, human neuroblastoma cells, human macrophage, and mouse macrophage.

I isolated total RNA from cells by using the Direct-zol™ RNA MiniPrep Kit from Zymo Research.  Then, I just needed to mix our isolated RNA with the iScript Supermix solution: one tube and one step. It only takes 40 min for the whole process from RNA to cDNA.

We have found that the iScript Supermix also increases data reproducibility by reducing the effect of pipetting variability and errors during set-up.  The Supermix also works across a broad linear dynamic range of total RNA input (1 µg–1 pg) and allows for detection of low-level target genes with real-time qPCR.