RASL‐seq for Massively Parallel and Quantitative Analysis of Gene Expression
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- Abstract
- Table of Contents
- Materials
- Figures
- 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
Table of Contents
- Introduction
- Strategic Planning
- Basic Protocol 1: The RASL‐seq Procedure
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
Materials
Basic Protocol 1: The RASL‐seq Procedure
Materials
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Figures
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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. View Image -
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. View Image
Videos
Literature Cited
| Literature Cited | |
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