Sep 20, 2017
Version 2
Bead Beating RNA extraction from 25 mm filter V.2
- Christa Smith1
- 1University of Georgia
- Moran Lab
Protocol Citation: Christa Smith 2017. Bead Beating RNA extraction from 25 mm filter. protocols.io https://dx.doi.org/10.17504/protocols.io.jx6cpre
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 20, 2017
Last Modified: March 22, 2018
Protocol Integer ID: 7902
Abstract
This protocol is for extracting RNA from 25 mm filters and can be used with filters stored in RNAlater for preservation. This protocol has been tested with 0.22 μm pore size Durapore 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).
Guidelines
Reference:
Satinsky BM, Gifford SM, Crump BC, Moran MA (2013) Use of internal standards for quantitative metatranscriptome and metagenome analysis. Meth Enzymol, ed DeLong EF (Academic, Burlington, MA), Vol 531, pp 237–250
Materials
MATERIALS
0.1 mm Zirconia/Silica BeadsBio Spec Products Inc.Catalog #11079101z
0.5 mm Zirconia/Silica BeadsBio Spec Products Inc.Catalog #11079105z
Glass beads, acid-washed, 425-600 μm (30-40 U.S. sieve) Merck MilliporeSigma (Sigma-Aldrich)Catalog #G8772-100G
Ambion Denaturation Solution Thermo ScientificCatalog #AM8540G
Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
RNeasy Mini KitQiagenCatalog #74104
TURBO DNA-free™ KitThermo ScientificCatalog #AM1907
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
STEP MATERIALS
Ambion Denaturation Solution Thermo ScientificCatalog #AM8540G
Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
RNeasy Mini KitQiagenCatalog #74104
TURBO DNA-free™ KitThermo ScientificCatalog #AM1907
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
Ambion Denaturation Solution Thermo ScientificCatalog #AM8540G
Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
RNeasy Mini KitQiagenCatalog #74104
TURBO DNA-free™ KitThermo ScientificCatalog #AM1907
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
Protocol materials
Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
TURBO DNA-free™ KitThermo ScientificCatalog #AM1907
RNeasy Mini KitQiagenCatalog #74104
Ambion Denaturation Solution Thermo ScientificCatalog #AM8540G
Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
RNeasy Mini KitQiagenCatalog #74104
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
TURBO DNA-free™ KitThermo ScientificCatalog #AM1907
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
RNeasy Mini KitQiagenCatalog #74104
TURBO DNA-free™ KitThermo ScientificCatalog #AM1907
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
0.1 mm Zirconia/Silica BeadsBio Spec Products Inc.Catalog #11079101z
0.5 mm Zirconia/Silica BeadsBio Spec Products Inc.Catalog #11079105z
Glass beads, acid-washed, 425-600 μm (30-40 U.S. sieve) Merck MilliporeSigma (Sigma-Aldrich)Catalog #G8772-100G
Ambion Denaturation Solution Thermo ScientificCatalog #AM8540G
Ambion Denaturation Solution Thermo ScientificCatalog #AM8540G
Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
Ambion Denaturation Solution Thermo ScientificCatalog #AM8540G
Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
RNeasy Mini KitQiagenCatalog #74104
TURBO DNA-free™ KitThermo ScientificCatalog #AM1907
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
Safety warnings
Proper sterile technique when handling samples and reagents is critical at every step to prevent introducing RNases that will degrade RNA samples.
Before start
Have the following on hand in addition to materials: forceps, 2.0 and 1.5 ml RNase free tubes, temperature controlled microcentrifuge, vortex with 2.0 ml adaptors, 21g1 needls, and 3 ml syringes.
Setup
Setup
Turn on 4°C centrifuge
Thaw internal standards on ice
Set up 2.0 ml tube adaptor on Mo Bio Vortex Genie 2.
Bead beating prep
Bead beating prep
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
400 µL
Protocol
NAME
3-bead mixtureCREATED BY
Christa Smith
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
1 mL
Ambion Denaturation Solution Thermo ScientificCatalog #AM8540G
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
Bead beating and RNA extraction
Bead beating and RNA extraction
Place sample tubes on vortex adaptor and beat for 5 min
00:05:00
Switch tube positions and beat another 5 min.
00:05:00
Centrifuge sample tubes for 1 min at 5000 rpm at 4°C
00:01:00
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
00:05:00
Transfer lysate to new 2.0 ml tube (Do not carry over any beads).
Add 1 volume of 100% ethanol to the lysate
1 mL
Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
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
RNeasy Mini KitQiagenCatalog #74104
Close tube gently and centrifuge 15 sec at 13000 rpm
00:00:15
Discard flow-through.
Repeat steps 16-18 until entire sample has been applied to column.
RNA purification
RNA purification
Add 700 μl Buffer RW1 to RNeasy Mini spin column.
Note
Here we are continuing to following Qiagen RNeasy Mini kit (step5 of Qiagen manual); finishing with two sequential elutions and spins of 35 μ
Centrifuge for 15 sec at 13000 rpm. Discard the flow-through.
00:00:15
Add 500 μl Buffer RPE to RNeasy spin column.
Centrifuge for 15 sec at 13000 rpm. Discard flow-through.
00:00:15
Add 500 μl Buffer RPE to RNeasy spin column.
Centrifuge for 2 min at 13000 rpm
00:02:00
Place column in new 2.0 ml collection tube.
Centrifuge for 1 min at 13000 rpm
00:01:00
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
00:01:00
Centrifuge for 1 min at 13000 rpm.
00:01:00
Add another 35 μl RNase-free water into same column/tube
Incubate for 1 min at room temperature
00:01:00
Centrifuge for 1 min at 13000 rpm
00:01:00
Discard column and place tube with RNA on ice
RNA quantification
RNA quantification
Quantify extracted RNA with Nanodrop (2 μl)
DNA removal
DNA removal
Mix the following in a 1.5 ml tube:
| component | amount | |
| extracted sample | 50 μl | |
| RNase-free water | 40 μl | |
| DNase reaction buffer | 10 μl | |
| Turbo DNase | 3 μl |
TURBO DNA-free™ KitThermo ScientificCatalog #AM1907
Incubate for 20 min at 37 °C
00:20:00
Add 3 μl more Turbo DNase to sample
Incubate an additional 20 min at 37 °C
00:20:00
Add 20 μl inactivation solution to sample (make sure solution is well mixed)
Vortex on and off for ~4 min
00:04:00
Spin for 1 min at 13000 rpm
00:01:00
Carefully, without disturbing inactivation reagent, remove supernatant (~100 μl) into a new 1.5 ml tube and place on ice
Quantification
Quantification
Quantify DNased RNA with Nanodrop (2 μl)
Optional
Optional
Optional: Clean and concentrate DNased RNA using Zymo RNA Clean & Concentrator-5 according to manufacturer protocol
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
Finish
Finish
Store RNA at -80 °C