1 Animal Genetics, Bioinformatics and Breeding, Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, Københavns Universitet2 IKVH Animal Genetics, Bioinformatics, and breeding, Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, Københavns Universitet3 Aalborg University4 Animal Genetics, Bioinformatics and Breeding, Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, Københavns Universitet5 Aalborg University
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.
Methods in Molecular Biology: Methods and Protocols, 2014, Vol 1182, p. 73-81