TITLE: RNA extraction and precipitation for 47 mm PES and polycarbonate filters
AUTHORS: Frank Ferrer-Gonzalez, Christa Smith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is a modified version of Bryn Durham et al., 2015 for extracting RNA from filters. </div><div class = "text-block">This protocol has been tested with 0.22 μm pore size Durapore filters and 2.0 μm polycarbonate filters. Custom synthesized RNA transcript standards are added at the time of extraction and are recovered post-sequencing for quantitative metatranscriptome analysis (Satinsky et al., 2013). To prevent RNA degradation, work in a clean environment with asceptic practices and avoid possible introduction of RNases to samples and reagents.</div></div>

[STEPS]
?. Prepare a 2.0 ml tube (low-binding) for each sample, with each tube containing 400 μl beads (mix of 200 μl 0.1 mm zirconium beads, 100 μl 0.5 mm glass beads, 100 μl 0.5 mm zirconium beads.
Glass beads are only needed when processing phytoplankton. If extracting from bacteria only then replace glass beads with more 0.5 mm zirconium.
?. Cut membrane filters using a RNAse free scissor.
?. Add T1E1 buffer with SDS 0.6%, Proteinase K 120 ng/μl.  Vortex for 10 minutes, incubate at RT for 50 minutes. Add 964 μl of T1E1 buffer, 30 μl of 20% SDS (20% SDS stock), and 6 μl of proteinase K, total 1000 μl.
T1E1 buffer: 10 mM Tris + 10 mM EDTA
?. Add the RNA standard - 0.5% of expected yield
?. Transfer supernatant to a new 2.0 ml microtube (low-binding). Be careful not to transfer the beads.
?. Centrifuge for 1 min at 8,000 rpm. If you have beads in your sample, repeat 5.
?. Add Acidic Phenol:Chloroform:Isoamyl alcohol (25:24:1) at a 1:1 volume. Shake until emulsified, don’t vortex.
?. Incubate the mixture on ice for 15 min.
?. Centrifuge at 13,400 rpm for 15 min, 4 Celsius. Collect supernatant in a new 2.0 ml tube.
?. Add 1 volume of Chloroform:Isoamyl alcohol (24:1), shake until emulsified.
Chloroform by itself will also work but it will create foam.
?. Incubate the mixture 15 min, on ice.
?. Centrifuge at 13,400 rpm for 15 min, 4 Celsius. Collect supernatant in a new tube.
?. Repeat Chloroform cleanup (steps 10, 11, and 12).
?. Add 1 volume of isopropanol.  Mix with a 21g syringe and shear DNA (8-10 times). Incubate at -20 Celsius overnight.
?. Centrifuge 30 min, 13,400 rpm, 4 Celsius. Remove supernatant, being careful not to lose the pellet.
You can transfer the supernatant to a new microtube, in case you lose the pellet you can recover it. This pellet almost always sticks to the microtube, the wash step is the one that mostly gets lose.
?. Add 1.0 ml 75% ethanol.
?. Centrifuge 10 min, 13,400 rpm, 4 Celsius. Discard supernatant, being careful not to lose the pellet.
?. Repeat ethanol wash (steps 16 and 17).
?. Short spin (~15s), remove remaining supernatant.
?. Repeat the short spin and make sure there is no ethanol in the sample.
If the pellet is wet it will look white, if it’s dry it will look clear.
?. Resuspend in ~30 ul RNAse free water.
This is depending on your experiment and needs, wait from 30 min to 1 hour on ice to take a reading, RNA takes time to dissolve in the sample.
?. Proceed to other applications or store at -80 Celsius.
?.