TITLE: Phenol-based RNA extraction from polycarbonate filters
AUTHORS: Robbie M. Martin, Steven W. Wilhelm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is an acid-phenol-based method for extracting RNA from samples collected onto polycarbonate filters.  Bead-beating is included to increase yields from difficult to lyse cells, for example, in some species of cyanobacteria.  We have used it successfully in RNA-sequencing projects involving lab cultures of cyanobacteria and from freshwater and marine environmental samples.</div></div>

[STEPS]
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Add 700 µL of Solution A (lysing buffer) to an MP Biomedical Lysing Matrix E tube (or equivalent bead beating tube).   Solution A consists of 0.5% SDS, 20 mM sodium acetate, and 10 mM EDTA in RNase-free molecular biology grade water.
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Pre-chill tube containing Solution A on ice to prevent tube from overheating during bead beating.
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Add sodium acetate to aqueous supernatant to a concentration of 0.3 M and mix.
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Add 2-2.5x volume of ice-cold 100% ethanol and mix well.
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Place in -80 °C freezer for at least 1 h, or preferably overnight.
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Unfold polycarbonate filter (either frozen, or thawed but maintained on ice) and place into pre-chilled bead-beating tube.
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Agitate tube on maximum speed for 40 s in Biospec Products Mini-beadbeater (or equivalent), then place on ice.
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Add 500 µL basic phenol with 8-hydroxyquinoline (pH 7.8-8.2) plus 100 µL chloroform to tube.
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Agitate tube on maximum speed for 40 s, then place briefly on ice to chill.
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Centrifuge tube at maximum speed in benchtop centrifuge for 5 min.
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Transfer as much of aqueous supernatant as possible from bead beating tube to new 2 mL centrifuge tube.
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Re-extract aqueous supernatant twice with 500 µL of acid phenol:chloroform:isoamyl alcohol (125:24:1; pH 4.8).
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Re-extract aqueous supernatant once with 500 µL basic phenol with 8-hydroxyquinoline plus 100 µL chloroform.
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Re-extract twice with 500 µL pure chloroform to remove last traces of residual phenol.
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Centrifuge at maximum speed at 0 °C for at least 30 min in refrigerated benchtop centrifuge.  If you expect low yields, or wish to increase recovery, spin for 1 h at maximum.
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Carefully decant ethanol supernatant without disturbing RNA pellet.
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Wash pellet by adding 1 mL of ice-cold 70% ethanol and gently pipetting to dislodge RNA pellet.
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Centrifuge at maximum speed at 0 °C for at least 15 min in a refrigerated benchtop centrifuge.
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Gently and carefully aspirate as much of the 70% ethanol from the tube as possible taking caution not to accidently aspirate the RNA pellet.
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Place the open tube in a 37 °C heating block for 3-5 min to evaporate all ethanol.  Do not dry pellet to completion, as this can make RNA difficult to redissolve in water.
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Dissolve the RNA pellet in an amount of RNase-free water appropriate for your expected yield and needed final concentration, typically about 50-100 µL.