TITLE: RNA extraction from Sterivex using phenol:chloroform
AUTHORS: Helena  Pound, Steven Wilhelm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This extraction protocol uses bead-beating techniques within the Sterivex to dislodge biomass from the Sterivex filter.  Biomass in solution is then removed from the Sterivex and RNA is extracted using acid phenol and chloroform.  The protocol has been tested and used on eutrophic freshwater samples dominated by toxic cyanobacteria (</span><span style = "font-style:italic;">Microcystis</span><span>).</span></div></div>

[STEPS]
?. [RNA extraction]
Remove luer-lock cap from Sterivex and carefully pour beads from Bashing Bead Lysis tube into the Sterivex
?. [RNA extraction]
Add 700 µL of solution A to Sterivex.
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Replace luer-lock cap and vortex for 20 min.
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Return Sterivex to ice, luer-lock side down to allow bubbles to dissipate.
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Use pliers to squeeze around the end of the Sterivex (opposite from the luer-lock). The Sterivex should pop apart and you can remove the end with the internal filter attached.
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Pipette all liquid from the Sterivex and put it back into the original Bashing Bead lysis tube (which should be sitting empty).
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This is challenging! A funnel helps if you can sterilize one. Save the BashingBead tube.
?. [RNA extraction]
Make solution A: 750 μL of 20% SDS, 600 μL of 0.5M EDTA, 200 μL of 3M sodium acetate, 28.45 mL of nuclease free water
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Make fresh weekly.
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Insert pipette past the neck of the Sterivex before ejecting liquid.
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If Sterivex don’t spin naturally on vortex, rotate the Sterivex 180° after 10 min and continue vortexing.
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Add 500 µL acid phenol:chloroform:IAA (125:24:1; pH 4.8) to the BashingBead tube.
?. [RNA extraction]
It's ok if you suck up some beads too.
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You should be pulling from the bottom layer of the thawed phenol:chloroform mixture.
?. [RNA extraction]
Beat on maximum speed for 40 s in bead beater and place on ice briefly to chill.
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Spin at maximum speed in a 4 °C benchtop centrifuge for 5 minutes.
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13,300 rpm for our centrifuge.
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Pull off as much of the aqueous supernatant as possible from the beating tube and place in a new 2 mL centrifuge tube.
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If there is a large white protein layer between your top and bottom layer, you may want to repeat the 500 µL acid phenol:chloroform:IAA addition, vortex, centrifuge, and add supernatant to new tube. You risk losing some RNA, but you can increase the quality.
?. [RNA extraction]
Add 500 μL chloroform to aqueous layer, vortex well, spin at max speed for 5 min.
?. [RNA extraction]
Repeat chloroform extraction to remove residual phenol.
?. [Ethanol Precipitation]
Add sodium acetate to ~0.3 M and mix.
?. [Ethanol Precipitation]
For 500 μL recovery, add 50 μL of 3M NaAc.
?. [Ethanol Precipitation]
Add 2-2.5x volumes of ice cold 100% ethanol and mix well.
?. [Ethanol Precipitation]
Typically fill the rest of the 1.5 mL microcentrifuge tube with ethanol.
?. [Ethanol Precipitation]
Place in -80 °C freezer for at least 1 hr.
?. [Ethanol Precipitation]
Can be stored at -80 °C overnight.
?. [RNA pellet wash]
Spin at maximum speed at 0 °C for at least 30 min in refrigerated centrifuge.
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If you suspect low yields, spin for 1 hr at max.This seems to help in RNA (or DNA) recovery.
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Carefully decant 100% ethanol without disturbing RNA (or DNA) pellet.
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Wash pellet by adding 1 mL of ice cold 70% ethanol and gently pipetting up and down enough to dislodge pellet.
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If you can see a pellet, you have at least ~2-5 µg nucleic acid. Do not panic if you cannot see pellet.
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You may not be able to see your pellet so be careful to avoid where it would be located.
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Spin at maximum speed at 0 °C for 15 minutes.
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Spin for 30 minutes if you expect low yields.
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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 pellet OR use a small pipette tip to suck out as much ethanol as you can.
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Place the open tube in a 37 °C heating block for about 3-5 min to evaporate all ethanol.
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Try to place heating block in sterile environemnt to avoid falling contamination, given that the tubes need to remain open during this step. It may take longer if you did not remove all excess ethanol in previous step.
?. [RNA pellet wash]
Dissolve the RNA (or DNA) pellet in the appropriate amount of RNAse/DNase-free water depending on expected yields and needed final concentration.