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Expression Profiling of ncRNAs Employing RNP Libraries and Custom LNA/DNA Microarray Analysis

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Regulatory RNAs

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Abstract

Recently, it has been shown by the ENCODE consortium that more than 90% of the human genome might be transcribed. While only about 1.5% of these transcripts correspond to mRNAs, it was proposed that the majority of them (i.e., 88.5%) might correspond to regulatory noncoding RNAs (ncRNAs). Numerous protocols dedicated to the generation of cDNA libraries coupled to next-generation sequencing (NGS) technologies are currently available to identify novel ncRNA species, and we have recently developed a novel procedure for the generation of ribonucleoprotein (RNP) libraries. To validate differential expression of ncRNAs identified using our or any library generation approach, we describe an innovative ncRNA profiling approach based on microarray technology. Employing LNA probes, dedicated to the analysis of small/microRNAs, and DNA probes, dedicated to the study of longer ncRNAs, our platform enables the study of most ncRNAs independently of their length in a single experiment. Detailed methodological solution description includes the automated design of probes to be spotted on the array, optimization of spotting and labeling of probes, as well as hybridization conditions. All the steps have been improved for the analysis of ncRNAs, which are generally difficult to study owing to their peculiarities in terms of secondary structure or abundance.

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Acknowledgments

Lorraine University and the Austrian Ministry of Science and Research (GEN-AU project consortium “noncoding RNAs” D-110420-011-015 to A.H. and M.S.) are acknowledged for financial support.

M.Z. was financed on a grant from the Austrian Ministry of Science and Research (D-110420-012-012).

K.S. was financed on a FWF grant (Project Number 2 F012060-03, Signal Processing in Neurons (SPIN) Ph.D. program to A.H.).

M.K. was financed on a GEN-AU grant from the Austrian Ministry of Science and Research (Project Number D-110420-011-015 to M.S.).

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Authors and Affiliations

  1. Section for Genomics and RNomics, Biocenter, Innsbruck Medical University, Innsbruck, Austria

    Konstantinia Skreka, Marek Zywicki, Alexander Hüttenhofer & Mathieu Rederstorff

  2. Institute for Genomics and Bioinformatics, Graz University of Technology, Graz, Austria

    Michael Karbiener & Marcel Scheideler

  3. Biopole, Lorraine University, CNRS UMR 7214, Vandoeuvre-les-Nancy, France

    Mathieu Rederstorff

Authors
  1. Konstantinia Skreka
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  2. Michael Karbiener
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  3. Marek Zywicki
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  4. Alexander Hüttenhofer
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  5. Marcel Scheideler
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  6. Mathieu Rederstorff
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Correspondence to Mathieu Rederstorff .

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  1. , New York State Department of Health, Wadsworth Center, New Scotland Avenue 120, New York, 12208, New York, USA

    Bibekanand Mallick

  2. A.J.C. Bose Centenary Building, P-1/12, CIT Scheme - VII M, Bose Institute, Kolkata, 700054, India

    Zhumur Ghosh

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Skreka, K., Karbiener, M., Zywicki, M., Hüttenhofer, A., Scheideler, M., Rederstorff, M. (2012). Expression Profiling of ncRNAs Employing RNP Libraries and Custom LNA/DNA Microarray Analysis. In: Mallick, B., Ghosh, Z. (eds) Regulatory RNAs. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-22517-8_9

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