Abstract
Quantitative PCR (qPCR) provides a robust method for quantifying DNA species. By combining modern qPCR techniques with the isolation of small RNA, the polyadenylation of the RNA, and the use of reverse transcriptase to create miRNA derived cDNA, it is now possible to use qPCR to quantify miRNA. This method is scalable and provides a useful addition to the retrovirologists’ toolbox. Here, we also describe the use of one-LTR infectious molecular clones to verify miRNA target sites within the retroviral LTR.
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References
Barik S (2005) Silence of the transcripts: RNA interference in medicine. J Mol Med 83:764–773
Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10:94–108
Umbach JL, Cullen BR (2009) The role of RNAi and microRNAs in animal virus replication and antiviral immunity. Genes Dev 23:1151–1164
Grassmann R, Jeang KT (2008) The roles of microRNAs in mammalian virus infection. Biochim Biophys Acta 1779:706–711
Klase Z, Jeang KT, Kashanchi F (2009) HIV-1 and RNA interference—examining a complex system. In: Yang D (ed) RNA viruses: host gene responses to infections. World Scientific Pub Co Inc., Singapore
Ouellet DL, Plante I, Barat C, Tremblay MJ, Provost P (2009) Emergence of a complex relationship between HIV-1 and the microRNA pathway. Methods Mol Biol 487:415–433
Huang J, Wang F, Argyris E, Chen K, Liang Z, Tian H, Huang W, Squires K, Verlinghieri G, Zhang H (2007) Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes. Nat Med 13:1241–1247
Schnettler E, de Vries W, Hemmes H, Haasnoot J, Kormelink R, Goldbach R, Berkhout B (2009) The NS3 protein of rice hoja blanca virus complements the RNAi suppressor function of HIV-1 Tat. EMBO Rep 10:258–263
Triboulet R, Mari B, Lin YL, Chable-Bessia C, Bennasser Y, Lebrigand K, Cardinaud B, Maurin T, Barbry P, Baillat V, Reynes J, Corbeau P, Jeang KT, Benkirane M (2007) Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Science 315:1579–1582
Abe M, Suzuki H, Nishitsuji H, Shida H, Takaku H (2010) Interaction of human T-cell lymphotropic virus type I Rex protein with Dicer suppresses RNAi silencing. FEBS Lett 584:4313–4318
Bellon M, Lepelletier Y, Hermine O, Nicot C (2009) Deregulation of microRNA involved in hematopoiesis and the immune response in HTLV-I adult T-cell leukemia. Blood 113:4914–4917
Tomita M, Tanaka Y, Mori N (2012) MicroRNA miR-146a is induced by HTLV-1 tax and increases the growth of HTLV-1-infected T-cells. Int J Cancer 130(10):2300–9
Bennasser Y, Le SY, Benkirane M, Jeang KT (2005) Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. Immunity 22:607–619
Klase Z, Kale P, Winograd R, Gupta MV, Heydarian M, Berro R, McCaffrey T, Kashanchi F (2007) HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR. BMC Mol Biol 8:63
Ouellet DL, Plante I, Landry P, Barat C, Janelle ME, Flamand L, Tremblay MJ, Provost P (2008) Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element. Nucleic Acids Res 36:2353–2365
Omoto S, Fujii YR (2006) Cloning and detection of HIV-1-encoded microRNA. Methods Mol Biol 342:255–265
Ying S-Y (2006) MicroRNA protocols. Humana Press, Totowa, NJ
Umbach JL, Cullen BR (2010) In-depth analysis of Kaposi’s sarcoma-associated herpesvirus microRNA expression provides insights into the mammalian microRNA-processing machinery. J Virol 84:695–703
Amen MA, Griffiths A (2011) Identification and expression analysis of herpes B virus-encoded small RNAs. J Virol 85:7296–7311
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233
Leonard J, Parrott C, Buckler-White AJ, Turner W, Ross EK, Martin MA, Rabson AB (1989) The NF-kappa B binding sites in the human immunodeficiency virus type 1 long terminal repeat are not required for virus infectivity. J Virol 63:4919–4924
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408
Acknowledgements
This work was supported by intramural funds from NIAID, NIH. We thank the members of the Jeang laboratory for critical reading of this writing.
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Klase, Z.A., Houzet, L., Jeang, KT. (2014). Quantification of miRNA by Poly(A)-RT-qPCR Arrays and Verification of Target Sites in HIV-1 Using a One-LTR Infectious Molecular Clone. In: Vicenzi, E., Poli, G. (eds) Human Retroviruses. Methods in Molecular Biology, vol 1087. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-670-2_23
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DOI: https://doi.org/10.1007/978-1-62703-670-2_23
Publisher Name: Humana Press, Totowa, NJ
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