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

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Abstract
Table of Contents
Materials
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Literature Cited

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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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
- MgCl₂
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

Figure 4.13.1 Overview of the RASL‐seq technology. All steps, including annealing, selection, ligation, and elution, can be carried out manually or on a customized Biomek FX robot. One of the targeting oligos (the upstream one) contains a 5′ phosphate. Each oligo contains a specific universal primer as indicated. After incorporating bar‐codes during PCR, the products are pooled, purified, and sequenced on an Illumina sequencer (GAII or HiSeq2000). The first sequencing run reads the ligated region from the P5 primer and the second identifies the bar‐coded region from the index primer.

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Figure 4.13.2 Reproducibility of RASL‐seq and similar fold‐differences recorded by different oligonucleotides targeting different regions in the same transcripts. (A ). Validation of androgen‐induced gene expression by RT‐qPCR on LNCaP cells. (B ) Similar fold‐differences despite distinct targeting efficiencies with different probe sets against the same transcripts.

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

Abstract

Table of Contents

Materials

Basic Protocol 1: The RASL‐seq Procedure

Figures

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Literature Cited