Total RNA was isolated from 10 to 5000 HeLa cells using RNeasy technology. One-tenth of each eluate (corresponding to RNA from 1 to 500 cells) was used for reverse transcription with Sensiscript Reverse Transcriptase. One-half of each RT reaction was then used in a 40-cycle PCR with QIAGEN Taq DNA Polymerase and primers specific for genes encoding β-actin, NF-κB, inositol-1,4,5-triphosphate receptor, or dystrophin. Sizes of the amplicons are as indicated.
The Sensiscript RT Kit includes dNTPs and an optimized reaction buffer that, together with the high affinity of Sensiscript Reverse Transcriptase for RNA, enables read-through of templates with high GC content or complex secondary structures. Due to this pre-optimization, tedious pipetting and pre-incubation steps are eliminated, and no additional RNase H digestion step is required.