Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

[Background]: MicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important biological role it is of great interest to quantitatively determine their expression level in different biological settings.

Results We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in microRNA quantification.

[Conclusions]: MiR-specific quantitative RT-PCR with DNA primers is a highly specific, sensitive and accurate method for microRNA quantification.

This work was supported by the Projects AGL2007-66371-C02-01 and AGL2010-22358-C02-01 and by the Consolider-Ingenio 2010 Program (CSD2007-00036) from Ministerio de Ciencia e Innovación. IB is recipient of PIF PhD fellowship from Universitat Autònoma de Barcelona.

Peer reviewed