RNA integrity and the effect on the real-time qRT-PCR performance Simone Fleige, Michael W. Pfaffl

Real time polymerase chain reaction is a very sensitive and powerful technique that is used to amplify a small amount of cDNA, reverse transcribed from mRNA, into large quantities to study low abundance gene expression. For successful and reliable gene expression data, it is important to start with intact RNA, since working with low quality RNA may strongly compromise the results. It is known that RNA is very sensitive to degradation due to many factors either from the extraction process or from inefficient lab management that may introduce exogenous contaminants to tissue samples. The RNA quality can be measured using different techniques, in this study Agilent Bioanalyzer was used, and RNA integrity number (RIN) that is higher than five was considered as good quality and higher than eight as perfect RNA quality.

In this review, the researchers compared the RNA quality of different bovine tissues and cell cultures to identify the influence of degraded RNA on the performance of qRT-PCR . In a study from Fleige and Pfaffl (2006) the purity and integrity of RNA samples derived from different bovine tissues and cell lines was measured using Bioanalyzer (2100). One distinct bovine tissue was degraded by enzymatic digest or with ultraviolet light to examine the effect of RNA integrity. The effect of RNA quality on RT-PCR performance was investigated by correlating RIN values with the cycle number(CP) of the PCR runs of four genes (18S,28S,B-Actin,IL-1B).  They found that a high quality RNA determined a lower CP than by less quality RNA, and there is significant correlation between RIN and CP that indicates with increasing RNA integrity the variability of RT-PCR results was decreased; however, after normalization of the CP by a reference gene (B-Actin) to decrease the RIN dependency, and by correlating RIN values with dCP values. They found that the normalized results showed minor influence of RNA quality on the expression results and the significant effect of RNA quality decreased to a minimum.

To get accurate RT-PCR results, it is important to start with high quality RNA; however, this review illustrates that moderate degraded samples may still give a reasonable data especially with the normalization; only the non-normalized values show a correlation between RNA quality and CP.

Posted by Wafa