Bead Beating RNA extraction from 25 mm filter
Turn on 4°C centrifuge. 
Thaw internal standards on ice. 
Set up 2.0 ml tube adaptor on Mo Bio Vortex Genie 2.
For each sample, prepare a 2.0 ml tube with the following 3-bead mixture: 200 μl 0.1 mm zirconium beads, 100 μl 0.4-0.6 mm glass beads, 100 μl 0.5 mm zirconium beads. 
Add Ambion Denaturation Solution to each tube. 
To each bead tube, add internal standards to reach ~0.5% of expected RNA yield. 
Place one 25 mm sample filter into each bead tube using RNase-free forceps. 
Place sample tubes on vortex adaptor and beat for 5 min. 
Switch tube positions and beat another 5 min.
Centrifuge sample tubes for 1 min at 5000 rpm at 4°C. 
Transfer as much of the RNA lysate as possible to a new 1.5 ml centrifuge tube (Can carryover some beads). 
Centrifuge RNA tubes for 5 min at 5000 rpm at 4°C. 
Transfer lysate to new 2.0 ml tube (Do not carry over any beads).
Add 1 volume of 100% ethanol to the lysate. 
Draw the RNA lysate up into a 3.0 ml syringe with a 21g1 gauge needle and pass it back out 8 times to shear RNA. 
Apply 700 μL of RNA lysate to RNeasy mini column. 
Close tube gently and centrifuge 15 sec at 13000 rpm. 
Discard flow-through.
Repeat steps 16-18 until entire sample has been applied to column.
Add 700 μl Buffer RW1 to RNeasy Mini spin column.
Centrifuge for 15 sec at 13000 rpm.
Discard the flow-through.
Add 500 μl Buffer RPE to RNeasy spin column.
Centrifuge for 15 sec at 13000 rpm.
Discard flow-through.
Add 500 μl Buffer RPE to RNeasy spin column.
Centrifuge for 2 min at 13000 rpm. 
Place column in new 2.0 ml collection tube.
Centrifuge for 1 min at 13000 rpm. 
Place column in a new 1.5 ml sterile tube. 
Add 35 μl RNase-free water directly onto filter and close the lid.
Incubate tube for 1 min at room temperature. 
Centrifuge for 1 min at 13000 rpm.
Add another 35 μl RNase-free water into same column/tube. 
Incubate for 1 min at room temperature. 
Centrifuge for 1 min at 13000 rpm. 
Discard column and place tube with RNA on ice. 
Quantify extracted RNA with Nanodrop (2 μl). 
Mix the following in a 1.5 ml tube:
componentamountextracted sample50 μlRNase-free water40 μlDNase reaction buffer10 μlTurbo DNase3 μl. 
Incubate for 20 min at 37 °C. 
Add 3 μl more Turbo DNase to sample. 
Incubate an additional 20 min at 37 °C. 
Add 20 μl inactivation solution to sample (make sure solution is well mixed).
Vortex on and off for ~4 min. 
Spin for 1 min at 13000 rpm. 
Carefully, without disturbing inactivation reagent, remove supernatant (~100 μl) into a new 1.5 ml tube and place on ice. 
Quantify DNased RNA with Nanodrop (2 μl) 
Optional: Clean and concentrate DNased RNA using Zymo RNA Clean & Concentrator-5 according to manufacturer protocol. 
Store RNA at -80 °C
