Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR

Abstract

Abstract Background The ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primer-independent cDNA synthesis using a popular RNase H- RT enzyme. A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs. Results Our findings on cellular RNA and in vitro synthesized RNA suggest that these products are likely the results of RNA self-priming to generate random cDNA products, which contributes to the loss of strand specificity. The use of RNase H+ RT enzyme and carrying the RT reaction at high temperature (50°C) greatly improved the strand specificity of the RT-PCR detection. Conclusion While RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when both sense and antisense RNA are expressed. For accurate interpretation of the results, it is essential to carry out the appropriate negative controls.