Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA

BMC Biotechnology

Rajiv Kumar Yadav¹  Florian Barbi¹  Antoine Ziller¹  Patricia Luis¹  Roland Marmeisse¹  M Sudhakara Reddy²  Laurence Fraissinet-Tachet¹ 

[1] Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université Lyon 1, Université de Lyon, Villeurbanne, France

[2] Department of Biotechnology, Thapar University, Patiala 147004, Punjab, India

关键词: Gel electrophoresis;    mRNA;    cDNA library;    cDNA;    Metatranscriptomics;   

Others  :  1084632
DOI  :  10.1186/1472-6750-14-80

 received in 2014-04-23, accepted in 2014-08-21,  发布年份 2014

【 摘 要 】

Background

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.

Results

We 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.

Conclusions

A 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.

【 授权许可】

   
2014 Yadav et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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