Preparation of mRNA for Expression Monitoring
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- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
Abstract
The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A+ ) versus total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.
Table of Contents
- Strategic Planning
- Basic Protocol 1: Amplification of mRNA for Expression Monitoring and Hybridization to Oligonucleotide Array Chips
- Support Protocol 1: In Vitro Transcription of Control Genes and Preparation of Transcript Pools
- Alternate Protocol 1: Solid‐Phase Reversible Immobilization (SPRI) Purification of cDNA and IVT Products
- Support Protocol 2: Quantitation of cDNA
- Reagents and Solutions
- Commentary
- Figures
- Tables
Materials
Basic Protocol 1: Amplification of mRNA for Expression Monitoring and Hybridization to Oligonucleotide Array Chips
Materials
Support Protocol 1: In Vitro Transcription of Control Genes and Preparation of Transcript Pools
Alternate Protocol 1: Solid‐Phase Reversible Immobilization (SPRI) Purification of cDNA and IVT Products
Materials
Support Protocol 2: Quantitation of cDNA
Materials
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Figures
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Figure 4.26.1 Chip analysis overview. Abbreviations: GIT, guanidine isothiocyanate, PE, phycoerythrin; SPRI, solid‐phase reversible immobilization. View Image
Videos
Literature Cited
Literature Cited | |
DeAngelis, M.M., Wang, D.G., and Hawkins, T.L. 1995. Solid‐phase reversible immobilization for the isolation of PCR products. Nucl. Acids Res. 23:4742‐4743. | |
Lockhart, D.J., Dong, H., Byrne, M.C., Follettie, M.T., Gallo, M.V., Chee, M.S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H., and Brown, E.L. 1996. Expression monitoring by hybridization to high‐density oligonucleotide arrays. Nature Biotechnol. 14:1675‐1680. | |
Schena, M., Shalon, D., Davis, R.W., and Brown, P.O. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467‐470. | |
Key References | |
Lockhart et al., 1996. See above. | |
This paper established that, in addition to its role as a resequencing tool, the oligonucleotide array could be used as a powerful methodology for transcriptional profiling. | |
Schena et al., 1995. See above. | |
A seminal paper on the potential of robotics and microfabrication to accelerate cDNA analysis using a highly parallel format. | |
Wodicka, L., Dong, H., Mittmann, M., Ho, M.H., and Lockhart, D.J. 1997. Genome‐wide expression monitoring in Saccharomyces cerevisiae. Nature Biotechnol. 15:1359‐1367. | |
Demonstration that oligonucleotide arrays can be used to simultaneously monitor expression of all genes of a eukaryotic organism. |