Abstract
This chapter describes two methods to measure expression of mature miRNA levels using qRT-PCR. The first method uses stem-loop RT primers to produce cDNA for specific miRNAs, a technique that our laboratory has modified to increase the number of miRNAs being reverse transcribed within a single RT reaction from one (as suggested by the manufacturer) to five. The second method uses a modified oligo(dT) technique to reverse transcribe all transcripts within an RNA sample; therefore, target miRNA and normalizing mRNA can be analyzed from the same RT reaction. We examined the level of miRNA-132, a miRNA known to be upregulated in granulosa cells following hCG treatment, using both of these methods. Data were normalized to GAPDH or snU6 and evaluated by ΔΔCt and standard curve analysis. There was no significant difference (P > 0.05) in miRNA-132 expression between the stem-loop and modified oligo(dT) RT methods indicating that both are statistically equivalent. However, from a technical point of view, the modified oligo(dT) method was less time consuming and required only a single RT reaction to reverse transcribe both miRNA and mRNA.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281-297
Griffiths-Jones S (2004) The microRNA registry. Nucleic Acids Res 32:D109-D111
Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34:D140-D144
Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:D154-D158
Song L, Tuan RS (2006) MicroRNAs and cell differentiation in mammalian development. Birth Defects Res C Embryo Today 78:140-149
Du T, Zamore PD (2005) microPrimer: the biogenesis and function of microRNA. Development 132:4645-4652
van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, Marshall WS, Hill JA, Olson EN (2008) Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci U S A 105:13027-13032
Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR (2005) MicroRNA expression profiles classify human cancers. Nature 435:834-838
Chan JA, Krichevsky AM, Kosik KS (2005) MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res 65:6029-6033
Lui WO, Pourmand N, Patterson BK, Fire A (2007) Patterns of known and novel small RNAs in human cervical cancer. Cancer Res 67:6031-6043
Kim VN (2004) MicroRNA precursors in motion: exportin-5 mediates their nuclear export. Trends Cell Biol 14:156-159
Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21:4663-4670
VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44:619-626
Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, 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:e179
Sharbati-Tehrani S, Kutz-Lohroff B, Bergbauer R, Scholven J, Einspanier R (2008) miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample. BMC Mol Biol 9:34
Lee DY, Deng Z, Wang CH, Yang BB (2007) MicroRNA-378 promotes cell survival, tumor growth, and angiogenesis by targeting SuFu and Fus-1 expression. Proc Natl Acad Sci U S A 104:20350-20355
Kunkel GR, Maser RL, Calvet JP, Pederson T (1986) U6 small nuclear RNA is transcribed by RNA polymerase III. Proc Natl Acad Sci U S A 83:8575-8579
Fiedler SD, Carletti MZ, Hong X, Christenson LK (2008) Hormonal regulation of MicroRNA expression in periovulatory mouse mural granulosa cells. Biol Reprod 79:1030-1037
Acknowledgments
We would like to express our most sincere appreciation to Stan Fernald for assistance with graphic design as well as Lacey Luense and Caitlin Healy for their critiques of this work.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Fiedler, S.D., Carletti, M.Z., Christenson, L.K. (2010). Quantitative RT-PCR Methods for Mature microRNA Expression Analysis. In: King, N. (eds) RT-PCR Protocols. Methods in Molecular Biology, vol 630. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-629-0_4
Download citation
DOI: https://doi.org/10.1007/978-1-60761-629-0_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-628-3
Online ISBN: 978-1-60761-629-0
eBook Packages: Springer Protocols