Ribonucleases (RNases) catalyze the degradation of RNA into smaller components, and are ubiquitous in a laboratory environment. While RNases have many essential functions within a cell, they are generally problematic outside the cell acting as a contaminant to researchers working with RNA. Endogenous RNases that co-purify during sample preparation and perspiration from ungloved hands can both contaminate pipet tips, tubes, buffers, water, and surfaces that come in contact with RNA. Researchers combat RNase contamination by using dedicated pipets, aerosol-barrier tips, DEPC-treated water and buffers, and wiping surfaces with RNase-inhibiting agents. Figure 1 shows varying levels of RNase contamination in different preparations of spleen and pancreas RNA. Contaminating RNase in a reverse transcription (RT) reaction can have adverse effects on gene expression analysis. Studies have shown that degraded RNA can cause threshold cycle (CT) delays in quantitative PCR (qPCR), which in turn can significantly affect overall mRNA quantification. Inaccurate representation of target gene levels can result from using degraded mRNA as a template for cDNA synthesis. The impact can be dramatic, especially when there are small differences in the expression levels between control and treated samples.
Not all cDNA synthesis kits have the same capacity to protect the integrity of RNA. An optimal level of RNase inhibitor is critical for powerful RNase inhibition and efficient reverse transcription. The iScript™ cDNA synthesis kit comes with a 5x iScript reaction mix and iScript reverse transcriptase, which is also blended with a potent RNase inhibitor. Figure 2 shows how the iScript cDNA synthesis kit significantly inhibits RNase activity.
To demonstrate the functional relevance of RNase inhibition in RT-qPCR, an experiment was performed with spiked RNaseA and appropriate controls (Figure 3).
There was no CT delay between cDNA synthesis performed without RNaseA and cDNA synthesis performed with spiked RNaseA (<0.5 CT difference), showing powerful inhibition of RNaseA. The iScript cDNA synthesis kit can inhibit contaminating RNases or RNases that co-purify with sample preparation, and allow accurate qPCR quantification of target genes. The two-tube format (5x reaction mix and iScript reverse transcriptase) also reduces the chance for RNaseA contamination during setup.
The benefit of powerful RNaseA inhibition is also offered by our 1-tube (iScript™ reverse transcription supermix for RT-qPCR) and high data throughput (iScript advanced cDNA synthesis kit for RT-qPCR) reverse transcription formats.
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