RASL‐seq for Massively Parallel and Quantitative Analysis of Gene Expression

Hairi Li1, Jinsong Qiu1, Xiang‐Dong Fu1

1 Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.13
DOI:  10.1002/0471142727.mb0413s98
Online Posting Date:  April, 2012
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Abstract

Large‐scale, quantitative analysis of gene expression can be accomplished by microarray or RNA‐seq analysis. While these methods are applicable to genome‐wide analysis, it is often desirable to quantify expression of a more limited set of genes in hundreds, thousands, or even tens of thousands of biological samples. For example, some studies may require monitoring a sizable panel of key genes under many different experimental conditions, during development, or following treatment with a large library of small molecules, for which current genome‐wide methods are either inefficient or cost‐prohibitive. This unit presents a method that permits quantitative profiling of several hundred selected genes in a large number of samples by coupling RNA‐mediated oligonucleotide Annealing, Selection, and Ligation with Next‐Gen sequencing (RASL‐seq). The method even allows direct analysis of RNA levels in cell lysates and is also adaptable to full automation, making it ideal for large‐scale analysis of multiple biological pathways or regulatory gene networks in the context of systematic genetic or chemical genetic perturbations. Curr. Protoc. Mol. Biol. 98:4.13.1‐4.13.9. © 2012 by John Wiley & Sons, Inc.

Keywords: gene expression; RNA‐mediated oligonucleotide; annealing; selection; RASL‐seq; bar‐coding strategies; high‐throughput screening

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: The RASL‐seq Procedure
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: The RASL‐seq Procedure

  Materials
  • Total RNA or cells from the cell type to be analyzed
  • RNase‐free water
  • MELT Total Nucleic Acid Isolation System for cell lysis (Ambion, cat. no. AM1983)
  • 2× binding cocktail (see recipe)
  • Washing buffer (see recipe)
  • T4 DNA ligase (Fermentas, cat. no. EL0011) containing 1× ligation buffer
  • Amplitaq Gold DNA polymerase (Life Technology, cat. no. N8080241) containing:
    • Amplitaq Gold PCR buffer
    • MgCl 2
  • PCR primers (see recipe for bar‐coding and P5 primer)
  • 10 mM dNTPs
  • NucleoSpin Extract II kit for PCR purification (Clontech, cat. no. 740609.50)
  • 384‐well plates
  • Thermal cycler (single or tetrad)
  • Magnetic stand
  • Qubit fluorometer (Life Technologies)
  • Illumina sequencer (ideally HiSeq2000)
  • Additional reagents and equipment for PCR (unit 15.1) and agarose gel electrophoresis (unit 2.5)
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Figures

Videos

Literature Cited

Literature Cited
   Fan, J.B., Yeakley, J.M., Bibikova, M., Chudin, E., Wickham, E., Chen, J., Doucet, D., Rigault, P., Zhang, B., Shen, R., McBride, C., Li, H.R., Fu, X.D., Oliphant, A., Barker, D.L., and Chee, M.S. 2004. A versatile assay for high‐throughput gene expression profiling on universal array matrices. Genome Res. 14:878‐885.
   Landegren, U., Kaiser, R., Sanders, J., and Hood, L. 1988. A ligase‐mediated gene detection technique. Science 241:1077‐1080.
   Li, H.R., Wang‐Rodriguez, J., Nair, T.M., Yeakley, J.M., Kwon, Y.S., Bibikova, M., Zheng, C., Zhou, L., Zhang, K., Downs, T., Fu, X.D., and Fan, J.B. 2006. Two‐dimensional transcriptome profiling: Identification of messenger RNA isoform signatures in prostate cancer from archived paraffin‐embedded cancer specimens. Cancer Res. 66:4079‐4088.
   Li, H., Zhou, H., Wang, D., Qiu, J., Zhou, Y., Li, X., Rosenfeld, M.G., Ding, S., and Fu, X.D. 2012. Versatile pathway‐centric approach based on high‐throughput sequencing to anticancer drug discovery. Proc. Natl. Acad. Sci. U.S.A. 2012 March 5 [Epub ahead of print].
   Wang, W., Barnaby, J.Y., Tada, Y., Li, H., Tor, M., Caldelari, D., Lee, D.U., Fu, X.D., and Dong, X. 2011. Timing of plant immune responses by a central circadian regulator. Nature 470:110‐114.
   Yeakley, J.M., Fan, J.B., Doucet, D., Luo, L., Wickham, E., Ye, Z., Chee, M.S., and Fu, X.D. 2002. Profiling alternative splicing on fiber‐optic arrays. Nat. Biotechnol. 20:353‐358.
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