SpringerProtocols: Abstract: Preparation of Full-Length cDNA Libraries: Focus on Metazoans

5. Preparation of Full-Length cDNA Libraries: Focus on Metazoans

By: Masako Harada¹, Yoshihide Hayashizaki^{1 2}

Abstract

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Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.

Affiliation(s): (1) Genome Exploration Research Group, Genomic Sciences Center (GSC), RIKEN, Yokohama, Japan
(2) Genome Science Laboratory, RIKEN, Wako, Japan

Book Title: Expressed Sequence Tags (ESTs): Generation and Analysis

Series: Methods in Molecular Biology | Volume: 533 | Year: 2009 | Page Range: 1-39 | DOI: 10.1007/978-1-60327-136-3_5

Subject: Genetics/Genomics

Key Words: mRNA - metazoan - full-length cDNA - CTAB-urea method - cap-trapper method - biotinylation of diol groups of mRNA - normalization and subtraction of cDNA

References

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