Stem‐Loop RT‐qPCR for miRNAs

Martha F. Kramer1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.10
DOI:  10.1002/0471142727.mb1510s95
Online Posting Date:  July, 2011
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Abstract

This unit presents a specific and sensitive quantitative reverse‐transcription PCR (RT‐qPCR) method for measuring individual microRNAs (miRNAs) in tissue or cultured cells. miRNAs are 17 to 24 nucleotides (nt) in length. Standard and quantitative PCR methods require a template that is at least two times the length of either of the specific forward or reverse primers, each typically ∼20 nt in length. Thus, the target minimum length is ≥40 nt, making miRNAs too short for standard RT‐qPCR methods. In this assay, each of the RT‐qPCR nucleic acid reagents, including the RT‐primer, the forward and reverse PCR primers, and the hydrolysis probe, contain design features that, together, optimize miRNA specificity and assay sensitivity. The RT‐primer contains a highly stable stem‐loop structure that lengthens the target cDNA. The forward PCR primer adds additional length with nucleotides that optimize its melting temperature (Tm) and enhance assay specificity. The reverse primer disrupts the stem loop. Assay specificity is further optimized by placement of the probe over much of the original miRNA sequence, and the probe Tm is optimized by addition of a minor groove binding (MGB) moiety. Curr. Protoc. Mol. Biol. 95:15.10.1‐15.10.15. © 2011 by John Wiley & Sons, Inc.

Keywords: miRNA; MIQE; RT‐qPCR

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Stem‐Loop RT‐qPCR for miRNAs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Stem‐Loop RT‐qPCR for miRNAs

  Materials
  • Fresh or frozen tissue (5 to 50 µg wet weight) and/or 106 to 107 cultured cells
  • mirVana miRNA isolation kit (Applied Biosystems, cat. no. AM1560) (also see units 4.2, 26.4, & 26.7)
  • flashPAGE Fractionator System (Applied Biosystems) (optional) containing:
    • flashPAGE Fractionator unit
    • Pre‐cast gels
    • Loading and running buffers
  • flashPAGE clean‐up kit (Applied Biosystems), optional
  • Nuclease‐free water
  • Nuclease‐free TE ( appendix 22)
  • 3 M sodium acetate, molecular biology grade ( appendix 22)
  • 100% ethanol, molecular biology grade
  • Quantification standards at 2.3 × 1010 molecules/µl (see recipe)
  • 5× (5.0 nM) stocks of stem‐loop primers (see recipe)
  • 20× stocks of combined forward primer, reverse primer, and probe (see recipe)
  • TaqMan MicroRNA RT kit (Applied Biosystems, cat. no. 4366596) containing:
    • 10× RT buffer
    • dNTP mix with dTTP (100 mM total)
    • RNase inhibitor (20 U/µl)
    • Multiscribe (MuLV) RT
  • TaqMan Universal PCR master mix (no AmpErase UNG; Applied Biosystems, cat. no. 4324018)
  • NanoDrop ND‐1000 or ND‐2000 (ThermoScientific) or comparable spectrophotometer
  • Refrigerated centrifuge
  • 0.6‐ and 1.5‐ml microcentrifuge (screw‐cap) tubes
  • 96‐well assay plates and sealing film
  • Miniplate Spinner mps 1000 (Labnet International)
  • Standard thermal cycler with 96‐well platform, optional
  • Optical 96‐well plates and optical sealing film specific for the instrument
  • Real‐time thermal cycler, e.g., Step‐One Plus (Applied Biosystems)
  • Microsoft Excel (or equivalent spreadsheet program)
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Figures

Videos

Literature Cited

   Burroughs, A.M., Ando, Y., de Hoon, M.J.4., Tomaru, Y., Nishibu, T., Ukekawa, R., Funakoshi, T., Kurokawa, T., Suzuki, H., Hayashizaki1, Y., and Daubl, C.O. 2010. A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness. Genome Res. 20:1398‐1410.
   Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., and Wittwer, C.T. 2009. The MIQE guidelines: Minimum information for publication of quantitative real‐time PCR experiments. Clinical Chemistry 55:1–12.
   Chen, C., Ridzon, D.A., Broomer, A.J., Zhou, Z., Lee, D.H., Nguyen, J.T., Barbisin, M., Xu, N.L., Mahuvakar, V.R., Andersen, M.R., Lao, K.Q., Livak, K.J., and Guegler, K.J. 2005. Real‐time quantification of microRNAs by stem‐loop RT‐PCR. Nucleic Acids Res. 33:e179.
   Cui, C., Griffiths, A., Li, G., Silva, L., Kramer, M.F., Gaasterland, T., Wang, X.J., and Coen, D.M. 2006. Prediction and identification of herpes simplex virus 1‐encoded microRNAs. J. Virol. 80:5499–5508.
   Cullen, B. 2004. Transcription and processing of human microRNA precursors. Mol. Cell 16:861–865.
   Ibrahim, F., Rymarquis, L.A., Kim, E.J., Becker, J., Balassa, E., Green, P.J., and Cerutti, H. 2010. Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas. Proc. Natl. Acad. Sci. U.S.A. 107:3906–3911.
   Jurak, I., Kramer, M.F., Mellor, J.C., van Lint, A.L., Roth, F.P., Knipe, D.M., and Coen, D.M. 2010. Numerous conserved and divergent microRNAs expressed by herpes simplex viruses 1 and 2. J. Virol. 84:4659‐4672.
   Logan, J., Edwards, K., and Saunders, N. 2009. Real‐Time PCR: Current Technology and Applications. Caister Academic Press, Norfolk, UK.
   Umbach, J.L., Kramer, M.F., Jurak, I., Karnowski, H.W., Coen, D.M., and Cullen, B.R. 2008. MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs. Nature 454:780–783.
Key References
  Bustin et al., 2009. See above.
  This seminal paper provides sensible and comprehensive guidelines for setting up, conducting, analyzing, and reporting the results of real‐time PCR experiments.
  Chen et al., 2005.
  This paper describes the stem‐loop approach and demonstrates the specificity of the stem‐loop primer (and coupled PCR reagents) for the mature miRNA and its precise sequence.
Internet Resources
  http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
  When an oligonucleotide sequence is inputted, this Internet tool returns the complement sequence, length, GC content, melting temperature, molecular weight, molecular extinction coefficient, nmol/OD260, and µg/OD260. This resource is very user‐friendly.
  http://bioinformatics.gene‐quantification.info
  This resource offers bioinformatical, biostatical as well as multi‐dimensional expression software tools, which are described well in Logan (), Chapter 5, Data Analysis Software, by Michael W. Pfaffl, Jo Vandesompele, and Mikael Kubista.
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