SpringerProtocols: Abstract: A Simple Method for Profiling miRNA Expression

A Simple Method for Profiling miRNA Expression

By: Jia-Wang Wang², Jin Q. Cheng²

Abstract

Full Text

Download PDF (307K)

Here we describe a simple protocol that uses positively charged nylon membrane dot blot to profile miRNA expression. A library of 515 antisense oligodeoxynucleotides of human and mouse mature miRNAs was synthesized and spotted on GeneScreen Plus membrane using a dot-blot equipment. Total RNA or enriched small molecular weight RNAs (smwRNAs) were enzymatically radiolabeled by poly (A) polymerase and then hybridized to the nylon membrane oligo arrays. The spot signal intensity on the membrane was analyzed using phosphorimaging. This method offers a convenient and economic way to simultaneously detect the expression of hundreds of miRNAs.

Affiliation(s): (2) H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL

Book Title: Apoptosis and Cancer: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 414 | Pub. Date: Oct-11-2007 | Page Range: 183-190 | DOI: 10.1007/978-1-59745-339-4_14

Subject: Cancer Research

Key Words: Dot blot - microRNA - hybridization - oligodeoxynucleotide library - phosphorimaging

References

Download References

1.	Lagos-Quintana, M., Rauhut, R., Lendeckel, W. and Tuschl, T. (2001) Identification of novel genes coding for small expressed RNAs. Science, 294, 853–858.

2.	Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116, 281–297.

3.	Berezikov, E., Guryev, V., van de Belt, J., Wienholds, E., Plasterk, R.H. and Cuppen, E. (2005) Phylogenetic shadowing and computational identification of human microRNA genes. Cell, 120, 21–24.

4.	Lim, L.P., Lau, N.C., Weinstein, E.G., Abdelhakim, A., Yekta, S., Rhoades, M.W., Burge, C.B. and Bartel, D.P. (2003) The microRNAs of Caenorhabditis elegans. Genes Dev, 17, 991–1008.

5.	Cullen, B.R. (2004) Transcription and processing of human microRNA precursors. Mol Cell, 16, 861–865.

6.	Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S. et al. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature, 425, 415–419.

7.	Gregory, R.I., Chendrimada, T.P., Cooch, N. and Shiekhattar, R. (2005) Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell, 123, 631–640.

8.	He, L. and Hannon, G.J. (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet, 5, 522–531.

9.	Lewis, B.P., Burge, C.B. and Bartel, D.P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 120, 15–20.

10.	Alvarez-Garcia, I. and Miska, E.A. (2005) MicroRNA functions in animal development and human disease. Development, 132, 4653–4662.

11.	Hall, P.A. and Russell, S.H. (2005) New perspectives on neoplasia and the RNA world. Hematol Oncol, 23, 49–53.

12.	Schutz, S. and Sarnow, P. (2006) Interaction of viruses with the mammalian RNA interference pathway. Virology, 344, 151–157.

13.	Shingara, J., Keiger, K., Shelton, J., Laosinchai-Wolf, W., Powers, P., Conrad, R., Brown, D. and Labourier, E. (2005) An optimized isolation and labeling platform for accurate microRNA expression profiling. RNA, 11, 1461–1470.

14.	Nelson, P.T., Baldwin, D.A., Scearce, L.M., Oberholtzer, J.C., Tobias, J.W. and Mourelatos, Z. (2004) Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods, 1, 155–161.

15.	Liu, C.G., Calin, G.A., Meloon, B., Gamliel, N., Sevignani, C., Ferracin, M., Dumitru, C.D., Shimizu, M., Zupo, S., Dono, M. et al. (2004) An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc Natl Acad Sci USA, 101, 9740–9744.

16.	Sioud, M. and Rosok, O. (2004) Profiling microRNA expression using sensitive cDNA probes and filter arrays. Biotechniques, 37, 574–576, 578–580.

17.	Krichevsky, A.M., King, K.S., Donahue, C.P., Khrapko, K. and Kosik, K.S. (2003) A microRNA array reveals extensive regulation of microRNAs during brain development. RNA, 9, 1274–1281.

Comments (Loading...)

Post comment/View all comments