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.
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 Equipment)
10-20 L Carboy (graduated)
Whirl Packs
Liquid nitrogen
RLT buffer (Qiagen)
Beta-mercaptoethanol
Setup:
Clean gloves should be worn whenever handling the filters or touching any of the interior components of the filter line. Forceps should be kept in clean, 50 ml conical tubes, preferably filled with ethanol. To prevent major changes in the transcript pool, keep filtering and sample handling times as short as possible.
Place one end of the tubing into the water at the desired depth for sampling. On the opposite end, attach the high volume 3 µm filter. Connect the 3 µm filter to the 0.22 µm filter tower. Direct the outflow from the 0.22 μm into a graduated carboy.
Procedure:
Part 2: Total RNA extraction from 0.22 μm filter
This part uses the QIAGEN RNeasy mini kit with modifications to the manufacturer’s instructions
Part 3. DNAse treatment
This part uses the Ambion TURBO DNA-free kit according to the manufacturer’s instructions
Part 4. First rRNA removal
This part uses the Epicentre mRNA-ONLY kit according to the manufacturer’s instructions
mRNA-ONLY 10X Reaction Buffer | 2.0 µl |
RNase Inhibitor | 0.5 µl |
Total RNA Sample (200 ng-10 µg) | 16.5 µl |
Terminator Exonuclease (1 Unit) | 1.0 µl |
Total volume | 20.0 µl |
Part 5. Second rRNA removal
This part uses the Ambion MICROBEnrich and MICROBExpress kits according to the manufacturer’s instructions. Both kits can be used multiple times to increase the efficiency of eukaryotic (MICROBEnrich) and prokaryotic (MICROBExpress) rRNA removal. The input of total RNA should be between 2-10 µg. The volume of RNA should be less than 15 µl. Thus, the ideal input is >150 ng/µl. –
MICROBEnrich
Part 6: mRNA Amplification
This part uses the Ambion MessageAmp II- Bacteria kit according to the manufacturer’s instructions. Calculate master mixes online at www.ambion.com/tools/ma2bact.
Part 7: cDNA synthesis
This part uses the Promega Universal RiboClone cDNA Synthesis System kit with slight modification to the manufacturer’s instructions. Standard input 2 µg; for 454 sequencing, it is recommended to begin with 10 µg. If the concentration is too low, the aRNA can be concentrated with either EtOH precipitation or vacuum concentration. If 10 µg is used as RNA input then scale all the reagents up by a factor of 5The enzymes come in units different each batch, thus volumes have to be calculated each time.
mRNA sample | 2 μg (or 10 μg if used for 454 sequencing) |
Random Hex Primer (0.5 mg/ml) | 2 μl |
Nuclease free water to volume | 0 (or non is sample was dilute) |
Total Volume | 15 μl |
Sample | 15 μl |
First Strand 5X Buffer | 5 μl |
RNasin Ribonuclease Inhibitor 40 u | 1 μl … 40 u/μl |
Total Volume | 21 μl |
Sample from step 1 | 21.0 μl |
Sodium Pyrophosphate, 40 mM | 2.5 μl |
AMV Reverse Trancriptase 30 u | 1.5 μl … 22 u/μl |
Nuclease free water | 0.0 μl |
Total Volume | 25.0 μl |
First-Strand reaction | 25.0 μl |
Second Strand 2.5X Buffer | 40.0 μl |
BSA, 1 mg/ml | 5.0 μl |
DNA Polymerase I 23 u | 3.0 μl |
RNase H 0.8 u | 0.5 μl … 1.5 u/μl |
Nuclease free water | 26.5 μl |
Total Volume | 100.0 μl |
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 contrast, environmental transcriptomics can be used to examine gene expression without constraints imposed by existing sequence data and with preference for those genes being actively expressed. Analysis of the mRNA pool in the environment can therefore provide one of the most effective ways of discovering connections between key activities and the organisms that mediate them.
The initial application of environmental transcriptomics provided one of the first views of the composition and dynamics of the bacterial mRNA pool in a natural ecosystem 3. Analysis of the expressed genes in transcript libraries from both a coastal salt marsh (Sapelo Island, GA) and an alkaline, hypersaline lake (Mono Lake CA) revealed gene sequences of biogeochemical interest, including environmental variants of several functional genes such as chitinases and sulfur oxidation genes that were specific to each of these ecosystems. It also provided evidence for novel, unexpected processes such as the microbial degradation of vascular plant- and algal-derived polyamines as a possible carbon and nitrogen sources.
As molecular techniques evolve to reduce the limitations associated with working with RNA from environmental samples and sequencing technologies improve, environmental transcriptomics has become a more widely used technique. It has been used to examine the functions of microorganisms in the surface ocean 6 and to compare day and night functional gene expression within the same environment 7. Environmental transcriptomics can also be used for gaining a better understanding of microbial responses to specific environmental factors and biogeochemistry. Genes or functions discovered using this technique can serve as targets for more quantitative studies of gene expression such as microarrays and quantitative PCR.
Funding was provided by The Gordon and Betty Moore Foundation grants and the National Science Foundation grant MCB-0702125.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
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 | 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 | C4360 | |
Wizard DNA Clean-Up System | kit | Promega | A7280 |