Advertisement

Extraction and qPCR-Based Detection of miRNAs from Cultured PBMCs of Bubaline Origin

  • Chandra S. MukhopadhyayEmail author
  • Ramneek Verma
  • Jasdeep Singh
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1656)

Abstract

MicroRNAs are small noncoding but functionally important RNA molecules that are involved in regulating diverse cellular, metabolic, and immune processes. Their small size necessitates modification in traditional acid phenol-chloroform based RNA isolation procedures to get highly enriched fraction of small RNA that includes miRNAs and siRNAs. Further, of the different methods available, real-time PCR is a powerful tool for precise and specific detection and quantification of miRNA. Moreover, real-time PCR is used to validate the screening or expression of miRNAs that are discovered during high-throughput sequencing, or microarray analysis. We demonstrate here the method of extraction of miRNAs from cultured PBMCs of bubaline origin followed by the qPCR-based (both SYBR green and TaqMan-based chemistries) identification of miRNAs expressed in response to TLR ligand stimulation.

Key words

Small RNAs PBMCs TaqMan MicroRNA assay miScript primer assay 

Notes

Acknowledgment

The work was supported by SERB-DST (Ministry of Science of Technology, Government of India) sponsored Research Grant SERC-LS-293-2011 (File Number SR/FT/LS-22/2011).

References

  1. 1.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefGoogle Scholar
  2. 2.
    Huntzinger E, Izaurralde E (2011) Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 12:99–110CrossRefGoogle Scholar
  3. 3.
    Baltimore D, Boldin MP, O’Connell RM, Rao DS, Taganov KD (2008) MicroRNAs: new regulators of immune cell development and function. Nat Immunol 9:839–845CrossRefGoogle Scholar
  4. 4.
    Ivey KN, Srivastava D (2010) MicroRNAs as regulators of differentiation and cell fate decisions. Cell Stem Cell 7:36–41CrossRefGoogle Scholar
  5. 5.
    Farazi TA, Hoell JI, Morozov P, Tuschl T (2013) MicroRNAs in human cancer. Adv Exp Med Biol 774:1–20CrossRefGoogle Scholar
  6. 6.
    Kolbert CP, Feddersen RM, Rakhshan F, Grill DE, Simon G, Middha S, Jang JS, Simon V, Schultz DA, Zschunke M, Lingle W, Carr JM, Thompson EA, Oberg AL, Eckloff BW, Wieben ED, Li P, Yang P, Jen J (2013) Multi-platform analysis of microRNA expression measurements in RNA from fresh frozen and FFPE tissues. PLoS One 8:e52517CrossRefGoogle Scholar
  7. 7.
    De Planell-Saguer M, Rodicio MC (2013) Detection methods for microRNAs in clinic practice. Clin Biochem 46:869–878CrossRefGoogle Scholar
  8. 8.
    Benes V, Castoldi M (2010) Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 50:244–249CrossRefGoogle Scholar
  9. 9.
    Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12. doi:10.1186/1746-4811-3-12. PMCID: PMC2225395CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, NL X, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179CrossRefGoogle Scholar
  11. 11.
    Singh J, Mukhopadhyay CS, Kaur S, Malhotra P, Sethi RS, Choudhary RK (2016) Identification of the MicroRNA repertoire in TLR-ligand challenged bubaline PBMCs as a model of bacterial and viral infection. PLoS One 11(6):e0156598. doi: 10.1371/journal.pone.0156598 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Chandra S. Mukhopadhyay
    • 1
    Email author
  • Ramneek Verma
    • 1
  • Jasdeep Singh
    • 2
  1. 1.School of Animal BiotechnologyGuru Angad Dev Veterinary and Animal Sciences University (GADVASU)LudhianaIndia
  2. 2.Department of Experimental Medicine and BiotechnologyPGIMERChandigarhIndia

Personalised recommendations