Abstract
MicroRNAs (miRNAs) represent a class of short (22 nt) noncoding RNAs that control gene expression post-transcriptionally. Microarray technology is frequently applied to monitor miRNA expression levels but is challenged by (i) the short length of miRNAs that offers little sequence for appending detection molecules; (ii) low copy number of some miRNA; and (iii) a wide range of predicted melting temperatures (Tm) versus their DNA complementary sequences. We recently developed a microarray platform for genome-wide profiling of miRNAs (miChip) by applying locked nucleic acid (LNA)-modified capture probes. Here, we provide detailed protocols for the generation of the miChip microarray platform, the preparation and fluorescent labeling of small RNA containing total RNA, its hybridization to the immobilized LNA-modified capture probes and the post-hybridization handling of the microarray. Starting from the intact tissue sample, the entire protocol takes ∼3 d to yield highly accurate and sensitive data about miRNA expression levels.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Lim, L.P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).
Farh, K.K. et al. The widespread impact of mammalian MicroRNAs on mRNA repression and evolution. Science 310, 1817–1821 (2005).
Ikeda, K. et al. Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody. J. Immunol. Methods 317, 38–44 (2006).
Gregory, R.I., Chendrimada, T.P., Cooch, N. & Shiekhattar, R. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123, 631–640 (2005).
Chen, C.Z., Li, L., Lodish, H.F. & Bartel, D.P. MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83–86 (2004).
Naguibneva, I. et al. The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation. Nat. Cell Biol. 8, 278–284 (2006).
Thompson, B.J. & Cohen, S.M. The Hippo pathway regulates the bantam microRNA to control cell proliferation and apoptosis in Drosophila. Cell 126, 767–774 (2006).
Cimmino, A. et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc. Natl. Acad. Sci. USA 102, 13944–13949 (2005).
Jopling, C.L., Yi, M., Lancaster, A.M., Lemon, S.M. & Sarnow, P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 309, 1577–1581 (2005).
Cai, X. et al. Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells. Proc. Natl. Acad. Sci. USA 102, 5570–5575 (2005).
Pfeffer, S. et al. Identification of microRNAs of the herpesvirus family. Nat. Methods 2, 269–276 (2005).
Bandres, E. et al. Identification by real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues. Mol. Cancer 5, 29 (2006).
Bottoni, A. et al. Identification of differentially expressed microRNAs by microarray: a possible role for microRNA genes in pituitary adenomas. J. Cell Physiol. 210, 370–377 (2007).
He, H. et al. The role of microRNA genes in papillary thyroid carcinoma. Proc. Natl. Acad. Sci. USA 102, 19075–19080 (2005).
Hossain, A., Kuo, M.T. & Saunders, G.F. Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA. Mol. Cell Biol. 26, 8191–8201 (2006).
Venturini, L. et al. Expression of the miR-17-92 polycistron in chronic myeloid leukemia (CML) CD34+ cells. Blood 109, 4399–4405 (2007).
Debernardi, S. et al. MicroRNA miR-181a correlates with morphological sub-class of acute myeloid leukaemia and the expression of its target genes in global genome-wide analysis. Leukemia 21, 912–916 (2007).
Sun, G., Li, H. & Rossi, J.J. Cloning and detecting signature MicroRNAs from mammalian cells. Methods Enzymol. 427, 123–138 (2007).
Griffiths-Jones, S. miRBase: the microRNA sequence database. Methods Mol. Biol. 342, 129–138 (2006).
Schmittgen, T.D., Jiang, J., Liu, Q. & Yang, L. A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res. 32, e43 (2004).
Goff, L.A. et al. Rational probe optimization and enhanced detection strategy for microRNAs using microarrays. RNA Biol. 2, 93–100 (2005).
Castoldi, M. et al. A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). RNA 12, 913–920 (2006).
Castoldi, M., Benes, V., Hentze, M.W. & Muckenthaler, M.U. miChip: a microarray platform for expression profiling of microRNAs based on locked nucleic acid (LNA) oligonucleotide capture probes. Methods 43, 146–152 (2007).
Vester, B. & Wengel, J. LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43, 13233–13241 (2004).
Valoczi, A. et al. Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res. 32, e175 (2004).
Miska, E.A. et al. Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol. 5, R68 (2004).
Fare, T.L. et al. Effects of atmospheric ozone on microarray data quality. Anal. Chem. 75, 4672–4675 (2003).
Chang, J. et al. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biol. 1, 106–113 (2004).
Acknowledgements
This work was supported by a Cancer Research Net grant (BMBF (NGFN) 201GS0450) to M.W.H. and M.U.M. Mirco Castoldi is supported by an Excellence Fellowship of The Medical Faculty of the University of Heidelberg.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Castoldi, M., Schmidt, S., Benes, V. et al. miChip: an array-based method for microRNA expression profiling using locked nucleic acid capture probes. Nat Protoc 3, 321–329 (2008). https://doi.org/10.1038/nprot.2008.4
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2008.4
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
