Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Summary

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

Abstract

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward ¹ and because of the relatively short half lives of mRNAs ². In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis ³. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation ⁴. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing ⁵. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression ⁶.

Protocol

Working with RNA

Because RNases are ubiquitous and mRNAs degrade rapidly, standard precautions for working in a ribonuclease-free environment must be followed and samples should be processed or preserved as soon as possible following collection.

Part 1: Environmental RNA Collection (designed to collect biomass in the 0.2- 3.0 µm size fraction)

Supplies needed:
Masterflex tubing
Peristaltic Pump
3 µm high volume pleated capsule filter
0.22 µm Supor or polycarbonate 142 mm filter
142 mm filter tower (Geotech Environmental....

Discussion

The investigation of gene expression by natural microbial communities has become common in recent years as a means to explore the ecological roles and functions of microorganisms. Used in combination with the detection, quantification, and characterization of marine microorganisms, analyses of gene expression can be used to link phylogeny to function in natural microbial communities. Many techniques for targeting functional gene expression rely on specific probes or primer sets designed for genes of known sequence. In co.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PowerSoil Bead Tubes	kit	MoBio	12866-25-PBT
RNeasy Mini Kit	kit	Qiagen	74104
TURBO DNA-free	kit	Ambion	AM1907
mRNA-ONLY Prokaryotic mRNA Isolation Kit	kit	Epicentre Biotechnologies	MOP51010/MOP51024
MICROBExpress Bacterial mRNA Enrichment Kit	kit	Ambion	AM1905
MICROBEnrich kit	kit	Ambion	AM1901
MessageAmp II-Bacteria RNA Amplification Kit	kit	Ambion	AM1790
Universal RiboClone cDNA Synthesis System	kit	Promega Corp.	C4360
Wizard DNA Clean-Up System	kit	Promega Corp.	A7280