Abstract
BackgroundConstruction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs.ResultsWe report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases.ConclusionsA simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.
Highlights
Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest
Eukaryotic cDNA synthesis from total soil RNA We extracted total RNA from three soil samples coming from contrasted geographic localities (Additional file 1)
We established a robust protocol to generate high quality eukaryotic sized cDNA libraries from total RNA extracted free from contaminations by either longer or shorter cDNAs
Summary
Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. The numerous eukaryotic microorganisms present in the environment potentially represent a rich source of genes encoding for novel enzymes or other proteins of interest in biotechnology. The numerous eukaryotic microorganisms present in the environment potentially represent a rich source of genes encoding for novel enzymes or other proteins of interest in biotechnology In this respect, functional metatranscriptomics has been demonstrated as a powerful tool in discovery of these genes [1,2,3,4,5,6]. This necessitates a highly efficient cDNA cloning approach which can make long eukaryotic transcripts available for functional studies
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