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The iScript RT-qPCR sample preparation reagent from Bio-Rad Laboratories provides rapid isolation of total RNA, enabling reverse transcription and real-time PCR to be performed directly from cell lysates. Scientists can use the protocol to efficiently remove genomic DNA and stabilize RNA in 5 min. Conventional RNA purification methods, such as organic extraction or column-based methods that include salting out or the use of magnetic particles, require numerous steps to purify RNA without contaminating genomic DNA and can take at least 30 min to complete. The reagent employs selective lysis of the cell membrane while leaving the nuclear membrane intact to achieve DNA-free RNA within 5 to 10 min. The single buffer formulation contains all the components needed to perform cell lysis, RNA stabilization, and genomic DNA removal for sensitive, quantitative gene expression analysis. It also offers sensitive detection of high-, medium- and low-copy gene targets directly from cell lysates. Multiplex real-time detection of up to four targets is also possible from as few as 10 cells. When cells are exposed to the reagent, cell disruption and lysis occurs in ~30 sec with the aid of mild vortexing. The reagent also stabilizes RNA and effectively inhibits intracellular ribonucleases (RNases). A short 2-min high-speed centrifugation yields total RNA that can be collected in the supernatant while the nuclei containing genomic DNA form a pellet and can be discarded. The resulting RNA preparation is ready for use, without further treatment, in subsequent reverse transcription and PCR/qPCR. RNA isolated with the reagent is stable for up to 6 months when stored at -20 C. The reagent is compatible with multiple PCR reagents already being used by researchers.
Tim Studt Editor in Chief