RNA degradation through RNase contamination causes many problems in downstream applications. RNA integrity is extremely important in gene expression studies where degradation can lead to misleading amounts of transcripts (1) (2). Preventing this degradation is essential and very simple using commercially available RNase inhibitors; however, some commercially available inhibitors can interfere with downstream applications and analyses after RNA isolation (1) (2).
The addition of as little as 1µl of Recombinant RNasin® Ribonuclease Inhibitor inhibits RNases and prevents degradation. Recombinant RNasin® Ribonuclease Inhibitor (Cat.# N2515) effectively protects RNA from RNase contamination in the laboratory (1) over a wide range of temperatures (4, 25, 37 and 50°C) and pH (5–8). Recombinant RNasin® Ribonuclease Inhibitor also offers a distinct advantage over native RNasin® Inhibitor because it is not isolated from human tissue, minimizing the risk of incorporating human nucleic acids into the assay (3).
Upstream gene expression analysis and RNA experiments commonly involve a workflow that includes RNA isolation, quantification, analysis of purity and integrity, and downstream applications such as RT-qPCR. Two common techniques used to quantitate RNA are absorbance using spectrophotometry and fluorescence using an RNA-specific binding dye. Spectrophotometry, using the NanoDrop® instrument, for example, is a simple, quick technique for quantification of RNA; however, it is not specific for RNA and results in inflated concentrations due to contaminants such as protein, DNA and organic compounds. The QuantiFluor™ RNA System (Cat.# E3310) is a fluorescent dye-based system that allows fast quantitation over a wide range of RNA concentrations with a lower limit of detection than the NanoDrop® instrument, making it an excellent quantitation system for samples with low RNA concentrations. The Agilent Bioanalyzer, which generates an RNA Integrity Number (RIN) to measure RNA integrity, also has become a popular method for analyzing RNA quality.
All techniques in this RNA workflow require intact RNA. Ideal RNase inhibitors protect against RNases but must not impair downstream assays. In this report, Recombinant RNasin® Ribonuclease Inhibitor was added to RNA samples collected using the Maxwell® 16 LEV simplyRNA Blood Kit (Cat. # AS1310). These samples were then tested using the NanoDrop® instrument, Quantifluor™ RNA System, Agilent Bioanalzyer and RT-qPCR before and after storage to determine the effect of Recombinant RNasin® Ribonuclease Inhibitor on downstream applications.