A water sample of 300 mL was added with an equal volume of RNA Later [ammonia sulfate 7.93 M, sodium citrate 0.025 M, EDTA 0.02 M qsp 1.5 L of RNAse free water (pH 5.2)], pre-filtered through 5 μm pore-size polycarbonate filters (Millipore) and then filtrated on 0.2 μm pore-size (pressure < 10 kPa) polycarbonate filters (Millipore) before storage at −80°C until nucleic acids extraction. It is well known that whatever the aquatic ecosystem studied, prefiltration allows some cells that are larger than their nominal pore sizes to pass through and can lead to the retention of smaller cells if the filters are clogged. However, after comparing nonfiltered and filtered fractions, in a previous study, we detected a slight decrease in total abundance (10–15%) but no modification of diversity inferred by morphological inspection (Lefranc et al., 2005 (link)). The nucleic acids extraction method was modified from Hugoni et al. (2013b (link)) using a combination of mechanical and enzymatic cell lysis, followed by extraction using the AllPrep DNA/RNA kit (Qiagen, Valencia, CA). RNA samples were tested for the presence of contaminating genomic DNA using PCR and then reverse transcribed with random primers using SuperScript® VILO (Invitrogen).
5 μm pore size polycarbonate filters
Manufactured by Merck Group
The 5 μm pore-size polycarbonate filters are a type of laboratory equipment used for filtration purposes. They are made of polycarbonate material and have a pore size of 5 micrometers. The core function of these filters is to separate particles or materials of a certain size from a liquid or gas sample.
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2 protocols using 5 μm pore size polycarbonate filters
1
Aquatic Microbial Nucleic Acids Extraction
2
Archaeal 16S rRNA Profiling from Environmental Samples
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A sub-sample (300 mL) added with an equal volume of RNA Later (ammonia sulfate 7.93 M, sodium citrate 0.025 M, EDTA 0.02 M, pH 5.2) was pre-filtered through 5-μm pore-size polycarbonate filters (Millipore) and collected on 0.2-μm pore-size (pressure <10 kPa) polycarbonate filters (Millipore) and stored at -80°C until nucleic acid extraction. The RNA extraction method was modified from Hugoni et al. [35] using a combination of mechanic and enzymatic cell lysis, followed by extraction using the AllPrep DNA/RNA kit (Qiagen, Valencia, CA). The RNA samples were tested for the presence of contaminating genomic DNA by PCR and then reverse transcribed with random primers using the SuperScript ® VILO (Invitrogen). The amplification of the V3-V5 region of the 16S rRNA genes was performed with universal archaeal primers Arch349F and Arch806R (Table 1, [38] ), followed by pyrosequencing using a Roche 454 GS-FLX system with titanium chemistry by a commercial laboratory (MR.DNA, Shallowater, TX, USA).
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