SuperScript™ First-Strand Synthesis System for RT-PCR From Invitrogen

Currently, I am utilizing the Invitrogen SuperScript™ First-Strand Synthesis System for cDNA synthesis from RNA isolated from different C. elegans mutant strains; the RNA was prepared using TRIzol Reagent. The SuperScript™ Kit comes with the necessary components to perform about 50 reactions. Each reaction can convert 1 ng – 5 ug of total RNA into first-strand cDNA. The components of this kit include oligo(dT), 10X RT buffer, MgCl₂, DTT, dNTP Mix, SuperScript™ II RNase H- Reverse Transcriptase (RT), RNaseOUT recombinant ribonuclease inhibitor, RNase H and DEPC-treated water. I have been using 2.5 ug of total RNA per reaction and I get about 20-40 ug of cDNA, as measured by spectrophotomety. I then use the cDNA to perform TaqMan® Real-Time PCR experiments.

The first-strand cDNA synthesis is catalyzed by SuperScript™ II RT, which has been engineered to eliminate RNase H activity, yielding greater full-length first-strand cDNA. SuperScript™ II RT is not inhibited by ribosomal RNA and transfer RNA, so it can effectively synthesize first-strand cDNA from a total RNA preparation. It also has increased thermal stability and so is effective up to 50^ºC. I prime my first-strand cDNA synthesis reaction using the oligo(dT) provided in the kit, which hybridizes to 3’ poly(A) tails. This method for priming first-strand synthesis is recommended when performing RT-PCR from a new mRNA target gene because oligo(dT) produces an RT-PCR product more consistently than other methods, such as random hexamers or gene-specific primers. The kit also provides RNase H, which removes the RNA template from the cDNA:RNA hybrid molecules after the first-strand synthesis. This increases sensitivity of PCR from cDNA. A control RNA is included in the kit to verify performance, but I have not been using it.

The manual that comes with the kit is very easy to follow. Basically, I mix my RNA with dNTP, Oligo(dT), and DEPC-treated water and incubate at 65^ºC for 5 min. Then, I add RT Buffer, MgCl₂, DTT and RNaseOUT, and incubate at 42^ºC for 2 min. Next, the SuperScript™ II RT is added and the reaction is incubated at 42^ºC for 50 min. The reaction is terminated after 15 min by placing the reaction at 70^ºC. RNase H is then added and the reaction is incubated for 20 min at 37^ºC before measuring the DNA concentration. The whole procedure is very quick, taking only a couple of hours.

I then use the cDNA in quantitative RT-PCR (qRT-PCR) assays (using the Chromo 4 from Bio-Rad Laboratories). We are interested in the function of Heat Shock Factor (HSF) as an activator in wild type and long-lived C. elegans mutants. The Invitrogen SuperScript™ II RT System allows me to make cDNA from my isolated total RNA quickly and efficiently. I get very good yields of cDNA and the easy-to-follow instruction manual makes this kit a great resource in our lab. I highly recommend this product.

Hee Chul Lee
Research Fellow
University of Michigan Medical School
Internal Medicine