Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Chen C, Ridzon DA, Broomer AJ et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179
Kumar P, Johnston BH, Kazakov SA (2011) miR-ID: a novel, circularization-based platform for detection of microRNAs. RNA 17:365–380
Shi R, Chiang VL (2005) Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 39:519–525
Chugh P, Dittmer DP (2012) Potential pitfalls in microRNA profiling, Wiley interdisciplinary reviews. RNA 3:601–616
Busk PK (2010) Method for quantification of small RNA species. WO2010085966(A2)
Balcells I, Cirera S, Busk PK (2011) Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers. BMC Biotechnol 11:70
Busk PK (2014) A tool for design of primers for microRNA-specific quantitative RT-qPCR. BMC Bioinformatics 15:29
SantaLucia J (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A 95:1460–1465
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Cirera, S., Busk, P.K. (2014). Quantification of miRNAs by a Simple and Specific qPCR Method. In: Alvarez, M., Nourbakhsh, M. (eds) RNA Mapping. Methods in Molecular Biology, vol 1182. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1062-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1062-5_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1061-8
Online ISBN: 978-1-4939-1062-5
eBook Packages: Springer Protocols