Abstract

AbstractThe objective of this publication is to provide the detailed protocol for metartancriptomisc studies of animal intestinal microbiota. The protocol describes isolation of high quality microbial community RNA from the mammalian intestinal content, subsequent mRNA enrichment, cDNA synthesis and sequencing. Twelve libraries were prepared, pooled in equimolar concentrations into a single library and sequenced on one GS Titanium 70 × 75 picotiter plate, following this protocol. The total number of reads obtained for 12 libraries was 1,155,062 (average 96,000 per library) and the combined size of 12 libraries was 521 million bases (average 43 million bases per library). The reported size of non-ribosomal RNA library fraction is ~15%, the fraction of non-ribosomal reads is ~17%. Hence we described a robust technique for metranscriptomic studies of animal intestinal microbiota. The double stranded cDNAs, prepared following this protocol, are suitable for pyrosequencing (454, Illumina), clone library construction or could be used to archive and store metaranscriptomic samples.

Highlights

  • One of the stated goals of the Human Microbiome Project is characterize bacterial “community genomes”, and determine their corresponding messenger RNAs, proteins and metabolic products (Turnbaugh et al, 2007)

  • The proposed protocol in this report describes in detail isolation of high quality microbial community RNA from the mammalian intestinal content, subsequent cDNA synthesis and sequencing

  • We have successfully utilized this protocol in pig animal model (Poroyko et al, 2010) using 454 Titanium pyrosequencing platform

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Summary

Introduction

One of the stated goals of the Human Microbiome Project is characterize bacterial “community genomes”, and determine their corresponding messenger RNAs, proteins and metabolic products (Turnbaugh et al, 2007). We have successfully utilized this protocol in pig animal model (Poroyko et al, 2010) using 454 Titanium pyrosequencing platform In this protocol we consider the importance of swift sample processing (Gilbert et al, 2008) to preserve the integrity of mRNA profiles. The total bacterial RNA isolation was performed using the “RiboPure-Bacteria Kit” (#AM1925; Ambion, TX). Sample harvesting Materials: portable Dewar flask, liquid nitrogen, 50ml sterile plastic centrifuge tubes, surgical equipment 1) Sacrifice the animal according to appropriate protocol. 2.3 RNA isolation Materials: portable Dewar flask, liquid nitrogen, Styrofoam container with dry ice, sterilized chilled pruner, handheld drill with 1/32 engraving cutter (Dremel), protective gaggles, Mini-Beadbeater-16 (BioSpec Products Inc., OK), RiboPure – Bacteria Kit (#AM1925; Ambion), dray bath 95 oC.

RNA precipitation Materials
Double-Stranded cDNA Synthesis Material
Conclusion
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