Quantification of miRNAs by a simple and specific qPCR method

TY - JOUR

T1 - Quantification of miRNAs by a simple and specific qPCR method

AU - Cirera Salicio, Susanna

AU - Busk, Peter K.

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

U2 - 10.1007/978-1-4939-1062-5_7

DO - 10.1007/978-1-4939-1062-5_7

M3 - Journal article

C2 - 25055902

SN - 1064-3745

VL - 1182

SP - 73

EP - 81

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

ER -