iScript cDNA Synthesis From Bio-Rad

The importance of the RNA world is increasingly appreciated. RNA molecules are not just inert messengers passing chromosomal information into proteins. They create a dynamically controlled network of intermediary regulatory factors. Because measurements of RNA molecules are done mainly after their conversion to complementary DNA (cDNA) molecules, reagents providing convenient and reliable reverse transcription of RNA to cDNA are of great value. Bio-Rad’s iScript cDNA Synthesis Kit comes right on board.

This kit is very easy to use. RNase H+ (more sensitive then RNase H-) is already pre-mixed with RNase inhibitor, while 5X Reaction Mix already contains oligo(dT)s, random hexamers and dNTPs. So you just need to add the Reaction Mix to your RNA sample, adjust total volume to 20 ul with nuclease-free water (provided), sprinkle it with a little bit of the enzyme (1 ul of RNase H+ per 20 ul reaction) and let the enzyme do its job for about 40 minutes (you can go have lunch). During that time, primers will anneal to the RNA (5 min, 25ºC), reverse transcriptase will produce cDNA (30 min, 42ºC), and RNA and cDNA will separate (5 min, 85ºC).

I have been successfully using Bio-Rad’s iScript cDNA Synthesis Kit for both end-point PCR and real-time PCR analyses of gene expression in mammalian brain tissue, primary cell lines and regular cell lines, even for transcripts longer then recommended cut-off of 1 kb. Its convenience and sensitivity are unsurpassed. Disadvantages could come from its strength, namely the premixture of oligo(dT)s and random hexamer primers. If you are interested in preparing cDNA of RNAs with poly-A tails only, then obviously this is not a kit for you. But Bio-Rad is prepared for a situation like that. Bio-Rad’s iScript Select cDNA Synthesis kit has both types of primers conveniently separated.