Generating a long DNA fragment of the target ncRNA for quantitative polymerase chain reaction by combining ncRNA-oligos hybridization and oligos ligation

Abstract

The poor reproducibility of the reverse transcription combined with quantitative polymerase chain reaction (RT-qPCR) results in an unacceptable reliability of publications based on these data. We established a novel method, in which two short complementary DNA oligos were hybridized with target ncRNA molecules and linked by DNA ligase to obtain a long DNA strand (HL-DNA) replacing cDNA for qPCR detection (HL-qPCR). A series of diluted samples prepared from the same total RNA resource were measured by HL-qPCR and RT-qPCR respectively to acquire their relative concentration of RNU4-1, AK026510 and SNORA73B. For every tested sample, the relative concentration of RNU4-1, AK026510 and SNORA73B obtained by HL-qPCR instead of RT-qPCR is closer to its corresponding true value without significant difference, demonstrating that HL-qPCR exhibits higher accuracy compared with RT-qPCR. With three independent repeats, no significant difference was observed among AK026510/RNU4-1 values of four samples diluted from the same RNA resource, by employing HL-qPCR but not RT-qPCR. It strongly suggests that the good reproducibility of HL-qPCR results from the stable efficiency of HL-DNA production regardless of the concentration and individual features of ncRNA. The novel HL-qPCR could be applied for the regular relative ncRNA concentration detection in the future.