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
