This kit is used for the reverse transcription (RT) of total or poly(A) RNA to first-strand cDNA using random hexamers, oligo-dT, or gene-specific primers. The cDNA generated can be used directly, without further purification, in many applications, such as PCR, real-time qPCR, arraying, and Northern blotting. The kit includes everything needed to make cDNA except your template RNA (1 pg to 5 ug of total RNA input is needed). The entire RT reaction can be performed in a PCR tube on a thermocycler in about 1.5 hours. The SuperScript III enzyme used with this kit is a version of MMLV reverse transcriptase that has reduced RNaseH activity, a longer half-life (220 minutes) and is more thermostable, resulting in increased specificity and higher yields. The first-strand cDNA obtained in the synthesis reaction may be used directly to amplify target cDNA by PCR or stored at -20ºC.
I use this kit routinely to synthesize cDNA from 100 ng-1 ug of rat heart total RNA; I then use the cDNA as template in real-time qPCR. Even if I don’t plan to use the cDNA right away, I will often convert my RNA to cDNA with this kit since cDNA is more stable than RNA. I then store the cDNA at -20ºC. In most of my experiments, I extract total RNA from rat hearts using Trizol, column-purify and DNase-treat the RNA, then convert it to cDNA using the random primers included with this kit (because I use 18S, which has no poly-A tail, as an endogenous control for relative quantification). I usually obtain at least 20 ug of cDNA no matter how much input total RNA I use. Following reverse transcription, I use 1-3 ug of the resulting cDNA template for use in real-time qPCR on an ABI 7000.