TRANSCRIPTOME 2002: From Functional Genomics to Systems
Specific Subtraction of Abundant mRNAs in Skeletal Muscle
Paolo Laveder, Cristiano De Pittą, Stefano Toppo, Giorgio Valle and Gerolamo Lanfranchi, CRIBI Biotechnology Center, University of Padova, ITALY
Through systematic sequencing of cDNA clones restricted to the 3'-end of each transcript (more than 35,000 clones analyzed), our group has built a catalogue of the most expressed genes in human skeletal muscle (http://muscle.cribi.unipd.it). A few exceptionally abundant (super-prevalent) mRNAs encoding myofibrillar and mitochondrial proteins accounted for about 40% of the total clones. Based on these data we have developed a new two-step strategy for producing subtracted cDNA libraries. The first subtractive step is limited only to the super-prevalent mRNAs and involves a novel use of oligonucleotide-directed RNase H digestion: DNA-oligonucleotides are selected informatically to digest the 3'UTRs of the target transcripts; mRNAs are obtained by in vitro transcription from 3'-end specific cDNA libraries. In the second step the hundred most abundant mRNA species are removed through subtractive hybridization. RNA drivers produced from 3'-end cDNA clones of our collection assured the maximum specificity of the hybridization reactions. Removal of mitochondrial transcripts required particular care, since they often are prematurely terminated. We showed that this technology allows a highly specific subtraction and that the resulting cDNA libraries are effectively enriched for genes expressed at low levels. In general, our subtractive approach would be appropriate for connective and epithelial tissues.
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