Abstract
Extracellular microRNAs (miRNAs) in body fluids have been identified as promising biomarkers for different human diseases. The high-throughput, multiplexed detection and quantification of these miRNAs are highly beneficial for the rapid and accurate diagnosis of diseases. Here, we developed a simple and convenient microarray-based technique, named ligase-assisted sandwich hybridization (LASH), for the detection and quantification of miRNAs. The LASH assay involves the hybridization of capture and detection probe pairs with the target miRNA to form a double-stranded structure which is then nick-sealed by T4 DNA ligase. Using this assay, we successfully demonstrated the multiplexed detection and quantification of different miRNAs in total RNA samples derived from blood obtained within 3 h. Here, we provide a detailed protocol for the LASH assay to detect a specific miRNA, as a model for the detection and quantification of extracellular miRNAs.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bushati N, Cohen SM (2007) microRNA functions. Annu Rev Cell Dev Biol 23:175–205
Sayed D, Abdellatif M (2011) MicroRNAs in development and disease. Physiol Rev 91:827–887
Iorio MV, Croce CM (2012) MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med 4:143–159
Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, Guo J, Zhang Y, Chen J, Guo X, Li Q, Li X, Wang W, Zhang Y, Wang J, Jiang X, Xiang Y, Xu C, Zheng P, Zhang J, Li R, Zhang H, Shang X, Gong T, Ning G, Wang J, Zen K, Zhang J, Zhang CY (2008) Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18:997–1006
Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O’Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 105:10513–10518
Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, Galas DJ, Wang K (2010) The microRNA spectrum in 12 body fluids. Clin Chem 56:1733–1741
Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, Tait JF, Tewari M (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 108:5003–5008
Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:423–433
Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659
Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, Yu L, Xiao T, Schafer J, Lee ML, Schmittgen TD, Nana-Sinkam SP, Jarjoura D, Marsh CB (2008) Detection of microRNA expression in human peripheral blood microvesicles. PLoS One 3:e3694
Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y, Ochiya T (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells. J Biol Chem 285:17442–17452
Cortez MA, Bueso-Ramos C, Ferdin J, Lopez-Berestein G, Sood AK, Calin GA (2011) MicroRNAs in body fluids—the mix of hormones and biomarkers. Nat Rev Clin Oncol 8:467–477
Ueno T, Funatsu T (2014) Label-free quantification of microRNAs using ligase-assisted sandwich hybridization on a DNA microarray. PLoS One 9:e90920
Southern EM, Mir KU, Shchepinov MS (1999) Molecular interactions on microarrays. Nat Genet 21:5–9
Chou CC, Chen CH, Lee TT, Peck K (2004) Optimization of probe length and the number of probes per gene for optimal microarray analysis of gene expression. Nucleic Acids Res 32, e99
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
Taylor DD, Gercel-Taylor C (2008) MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 110:13–21
Li A, Omura N, Hong SM, Vincent A, Walter K, Griffith M, Borges M, Goggins M (2010) Pancreatic cancers epigenetically silence SIP1 and hypomethylate and overexpress miR-200a/200b in association with elevated circulating miR-200a and miR-200b levels. Cancer Res 70:5226–5237
Bullard DR, Bowater RP (2006) Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochem J 398:135–144
Lohman GJ, Tabor S, Nichols NM (2011) DNA ligases. Curr Protoc Mol Biol 94:3.14.1–3.14.7
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
Acknowledgments
This research was partly supported by the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) from the Japan Society for the Promotion of Science (JSPS) and by the Center of Innovation Program from Japan Science and Technology Agency (JST).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Iizuka, R., Ueno, T., Funatsu, T. (2016). Detection and Quantification of MicroRNAs by Ligase-Assisted Sandwich Hybridization on a Microarray. In: Li, P., Sedighi, A., Wang, L. (eds) Microarray Technology. Methods in Molecular Biology, vol 1368. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3136-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3136-1_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3135-4
Online ISBN: 978-1-4939-3136-1
eBook Packages: Springer Protocols