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


CONCLUSIONS
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.
Davis
Claytus
C
Department of Virology and Developmental Genetics, Faculty of Health Science, Ben Gurion University of the Negev, Beer Sheva, Israel. clay@bgu.ac.il
Barvish
Zeev
Z
Gitelman
Inna
I
D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D020869:Gene Expression Profiling; D015723:Gene Library; D006868:Hydrolysis; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA; D004262:DNA Restriction Enzymes