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Quantitative RT-PCR for MicroRNAs in Biofluids

  • Michael Thorsen
  • Thorarinn Blondal
  • Peter MouritzenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1641)

Abstract

MicroRNAs in biofluids hold great promise as minimally invasive diagnostic biomarkers for a wide range of diseases and biological processes. One of the most sensitive technologies for detection and measuring expression levels of microRNA is quantitative RT-PCR. However, quantification of microRNA in biofluid samples is challenging in many ways. Biofluids contain low levels of RNA and high levels of inhibitors of enzymatic processes like reverse transcription and PCR. Furthermore, biofluids are susceptible to many preanalytical variables. Here we describe procedures developed to address these challenges, which include highly sensitive and accurate microRNA detection methods, combined with optimized protocols for sample handling and preparation, and extensive quality control (QC) procedures.

Key words

Biofluids RNA isolation microRNA RT-qPCR Expression analysis 

References

  1. 1.
    Jacobsen N, Andreasen D, Mouritzen P (2011) Profiling MicroRNAs by real-time PCR. MicroRNAs in development volume 732 of the series. Methods Mol Biol 732:39–54CrossRefPubMedGoogle Scholar
  2. 2.
    Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5(10):R80CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Tichopad A, Kitchen R, Riedmaier I, Becker C, Ståhlberg A, Kubista M (2009) Design and optimization of reverse-transcription quantitative PCR experiments. Clin Chem 55(10):1816–1823CrossRefPubMedGoogle Scholar
  4. 4.
    Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B 57:289–300Google Scholar
  5. 5.
    Dunn OJ (1959) Estimation of the medians for dependent variables. Ann Math Stat 30(1):192–197CrossRefGoogle Scholar
  6. 6.
  7. 7.
    Blondal T, Jensby Nielsen S, Baker A, Andreasen D, Mouritzen P, Wrang Teilum M, Dahlsveen IK (2013) Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods 59(1):S1–S6Google Scholar
  8. 8.
    Merkerova M, Belickova M, Bruchova H (2008) Differential expression of microRNAs in hematopoietic cell lineages. Eur J Haematol 81(4):304–310Google Scholar
  9. 9.
    Ruijter JM, Pfaffl MW, Zhao S, Spiess AN, Boggy G, Blom J, Rutledge RG, Sisti D, Lievens A, De Preter K, Derveaux S, Hellemans J, Vandesompele J (2013) Evaluation of qPCR curve analysis methods for reliable biomarker discovery: Bias, resolution, precision, and implications. Methods 59(1):32–46Google Scholar
  10. 10.
    Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F, Speleman F, Vandesompele J (2009) A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol 10(6):R64CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Pritchard CC, Cheng HH, Tewari M (2012) MicroRNA profiling: approaches and considerations. Nat Rev Genet 13(5):358–369CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Andreasen D, Fog JU, Biggs W, Salomon J, Dahslveen IK, Baker A, Mouritzen P (2010) Improved microRNA quantification in total RNA from clinical samples. Methods 50(4):S6–S9CrossRefPubMedGoogle Scholar
  13. 13.
    Kim YK, Yeo J, Kim B, Ha M, Kim VN (2012) Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells. Mol Cell 46(6):893–895CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Michael Thorsen
    • 1
  • Thorarinn Blondal
    • 1
  • Peter Mouritzen
    • 1
    Email author
  1. 1.Exiqon A/SVedbækDenmark

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