Circumventing qPCR inhibition to amplify miRNAs in plasma

Circulating microRNAs (c-miRNAs) have be identified in saliva, urine and blood, which has led to increasing interest in their development as biomarkers for diverse diseases including cancers. One of the key advantages of c-miRNAs over other biomarkers is the ability to be amplified and quantified by quantitative PCR (qPCR). However, at phlebotomy when whole blood is dispensed into heparinized tubes, residual levels of the anti-coagulant lithium heparin may remain in the plasma and hence with RNA isolated from the plasma. This can confound the detection of c-miRNAs by qPCR because it inhibits reverse transcriptase (RT). Here we present a procedure, modified from earlier techniques, to detect c-miRNAs in plasma that improves sensitivity and streamlines performance.

Treatment of total RNA isolated from human blood plasma with Bacteroides heparinase I during reverse transcription at 37°C for one hour improved sensitivity and performance of the qPCR. This is in comparison to no treatment or treatment of the RNA prior to RT, which is the current suggested method and exposes plasma to Flavobacterium heparinum heparinase I for up to 2 hours before RT. This modest alteration improved qPCR performance and resulted in lowered threshold cycles (Ct) for detection of the target sequence, candidate c-miRNA biomarkers, and controls. It also reduced the expense and number of processing steps, shortening the duration of the assay and minimizing exposure of RNA to elevated temperatures.

Incorporating Bacteroides heparinase I treatment into conventional RT protocols targeting c-miRNA in plasma can be expected to expedite the discovery of biomarkers.