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Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues

Peltier, Heidi J., Latham, Gary J.·2008·RNA
DOI 10.1261/rna.939908
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Abstract

Proper normalization is a critical but often an underappreciated aspect of quantitative gene expression analysis. This study describes the identification and characterization of appropriate reference RNA targets for the normalization of microRNA (miRNA) quantitative RT-PCR data. miRNA microarray data from dozens of normal and disease human tissues revealed ubiquitous and stably expressed normalization candidates for evaluation by qRT-PCR. miR-191 and miR-103, among others, were found to be highly consistent in their expression across 13 normal tissues and five pair of distinct tumor/normal adjacent tissues. These miRNAs were statistically superior to the most commonly used reference RNAs used in miRNA qRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA. The most stable normalizers were also highly conserved across flash-frozen and formalin-fixed paraffin-embedded lung cancer tumor/NAT sample sets, resulting in the confirmation of one well-documented oncomir (let-7a), as well as the identification of novel oncomirs. These findings constitute the first report describing the rigorous normalization of miRNA qRT-PCR data and have important implications for proper experimental design and accurate data interpretation.

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  • A subset of miR-15/107 miRNA genes has been detected in mammalian tissues including heart, skeletal muscle, brain, lung, liver, kidney, spleen and placenta.

    “A subset of these miRNA genes has been detected in mammalian tissues including heart, skeletal muscle, brain, lung, liver, kidney, spleen and placenta [25–31].”

    From Expression of miR-15/107 Family MicroRNAs in Human Tissues and Cultured Rat Brain Cells (2014) · cites this paper as [31]

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Research works printed but not linked: 7

  1. Doleshal, M., Magotra, A.A., Choudhury, B., Cannon, B.D., Labourier, E., and Szafranska, A.E. 2008. Evaluation and valida- tion of total RNA extraction methods for microRNA expression analyses in formalin-fixed, paraffin-embedded tissues. J. Mol. Diag. (in press).
  2. Liang, Y., Ridzon, D., Wong, L., and Chen, C. 2007. Characterization of microRNA expression profiles in normal human tissues. BMC Genomics 8: 166; doi: 10.1186/1471-2164-8-166. · DOI
  3. Mattie, M.D., Benz, C.C., Bowers, J., Sensinger, K., Wong, L., Scott, G.K., Fedele, V., Ginzinger, D., Getts, R., and Haqq, C. 2006. Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies. Mol. Cancer 5: 24; doi: 10.1186/1476- 4598-5-24. · DOI
  4. Shell, S., Park, S.M., Radjabi, A.R., Schickel, R., Kistner, E.O., Jewell, D.A., Feig, C., Lengyel, E., and Peter, M.E. 2007. Let-7 expression defines two differentiation stages of cancer. Proc. Natl. Acad. Sci. 104: 11400-11405.
  5. Szabo, A., Perou, C.M., Karaca, M., Perreard, L., Quackenbush, J.F., and Bernard, P.S. 2004. Statistical modeling for selecting house- keeper genes. Genome Biol. 5: R59; doi: 10.1186/gb-2004-5-8-r59. · DOI
  6. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of mul- tiple internal control genes. Genome Biol. 3: RESEARCH0034.1; doi: 10.1186/gb-2007-3-8-research0034. · DOI
  7. Volinia, S., Calin, G.A., Liu, C.G., Ambs, S., Cimmino, A., Petrocca, F., Visone, R., Iorio, M., Roldo, C., Ferracin, M., et al. 2006. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl. Acad. Sci. 103: 2257-2261.

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Authors
Peltier, H. J., & Latham, G. J.
Venue
RNA
Publisher
Cold Spring Harbor Laboratory
Published
2008-03-28
Volume
14
Issue
5
Pages
844-852
ISSN
  • 1355-8382
  • 1469-9001
DOI
10.1261/rna.939908
Cited by
716
References
1
Language
en

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