A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA.

A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA. Research Abstract Details 

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A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA. Abstract Text:

Claytus Davis,Zeev Barvish,Inna Gitelman,Claytus Davis,Zeev Barvish,Inna Gitelman,Claytus Davis,Zeev Barvish,Inna Gitelman,

ABSTRACT: BACKGROUND: The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. RESULTS: We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed) total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. CONCLUSION: We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.

A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA. Publishing Authors By Initials

C Davis,Z Barvish,I Gitelman,C Davis,Z Barvish,I Gitelman,C Davis,Z Barvish,I Gitelman,

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A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: BMC genomics

VOLUME: 8

Page Numbers: 363

Journal Abbreviation: BMC Genomics

ISSN: 1471-2164

DAY: 9

MONTH: 10

YEAR: 2007

A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA. Information

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LANGUAGE: eng

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AFFILIATION: Department of Virology and Developmental Genetics, Faculty of Health Science, Ben Gurion University of the Negev, Beer Sheva, Israel. clay@bgu.ac.il.

Country: England

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MEDLINETA: BMC Genomics

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