Quantification of microRNAs by a simple and specific qPCR method

MicroRNAs are powerful regulators of gene expression at post-transcriptional level and play important roles in many biological processes and in disease. The rapid pace of the emerging field of microRNAs has open new avenues for development of techniques to quantitatively determine microRNA expression levels in different systems.In this chapter 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. This method quantifies synthetic templates over eight orders of magnitude and successfully discriminate microRNAs that differ by one single nucleotide. Due to the usage of DNA primers this method allows higher amplification efficiencies than a similar method based on locked nucleic acids-spiked primers. The high efficiency translates into higher sensitivity and precision in microRNA quantification. Furthermore, the method is easy to perform with common laboratory reagents, which allows microRNA quantification at low cost. MicroRNAs (miRNAs) are powerful regulators of gene expression at posttranscriptional level and play important roles in many biological processes and in disease. The rapid pace of the emerging field of miRNAs has opened new avenues for development of techniques to quantitatively determine miRNA expression levels in different systems. In this chapter we describe a PCR method for quantification of miRNAs based on a single reverse transcription reaction for all miRNAs combined with real-time PCR with two miRNA-specific DNA primers. This method quantifies synthetic templates over eight orders of magnitude and successfully discriminates miRNAs that differ by one single nucleotide. Due to the usage of DNA primers this method allows higher amplification efficiencies than a similar method based on locked nucleic acid-spiked primers. The high efficiency translates into higher sensitivity and precision in miRNA quantification. Furthermore, the method is easy to perform with common laboratory reagents, which allows miRNA quantification at low cost.