Jun 23, 2017

Public workspaceRNA Extraction from Sterivex filters

DOI
dx.doi.org/10.17504/protocols.io.gmkbu4w
  • Lauren Krausfeldt
  • The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Lauren Krausfeldt
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DOI: dx.doi.org/10.17504/protocols.io.gmkbu4w
External link: https://mobio.com/media/wysiwyg/pdfs/protocols/14600-S.pdf
Protocol Citation: Lauren Krausfeldt 2017. RNA Extraction from Sterivex filters . protocols.io https://dx.doi.org/10.17504/protocols.io.gmkbu4w
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: December 01, 2016
Last Modified: March 27, 2018
Protocol Integer ID: 4492
Keywords: RNA Extraction, MoBio, Sterivex, Qiagen
Abstract
RNA extraction from water collected on Sterivex. The DNeasy PowerWater Sterivex kit is used and modified specifically for RNA. Also includes modifications for samples preserved in RNAlater.
Guidelines
What you will need:
-Kit: Qiagen DNeasy PowerWater Sterivex kit (formerly MoBio PowerWater DNA Isolation for Sterivex (Catalog# 14600-50-NF))
-Kit: Invitrogen Turbo DNase (Catalog# AM1907)
-B-mercaptoethanol (BME) *
-65ºC incubator
-70ºC incubator
-4ºC refrigerator
-Vortex
-100% RNase free EtOH
-RNase free H2O
-60 mL or 3mL sterile syringes (If using RNAlater solution)
* See warnings
Safety warnings
-WORK IN FUME HOOD FOR ALL STEPS INVOLVING  B-Mercaptoethanol (BME)
https://www.sciencelab.com/msds.php?msdsId=9924612
-Wear gloves and avoid skin contact with all reagents in MoBio and Invitrogen kits.
Before start
This protocol is modified from a MoBio Protocol. You will need:
a.) MoBio PowerWater DNA Isolation for Sterivex Kit (Catalog# 14600-50-NF) 
b.) Invitrogen Turbo DNase (Catalog # AM1907)
Preparation
Preparation
Add solution ST1A to bottle ST1B  (provided in kit).
Note
Only required the first time you use ST1B, it can be kept at 4°C for storage when not in use.
Warm solutions ST2 and ST4 prior to use at 65°C for 5-10 minutes. Use while warm.
Note
Solutions ST2 and ST4 are salty solutions and precipitates, must be dissolved prior to use.
 If Sterivex were capped with clay at the outlet and no screw cap is available, use electrical tape to secure the opening. The clay may not hold during the entire extraction process and this will prevent your lysate from leaking. 
(900μL) ST1B/Beta-mercaptoethanol (BME) solution will be needed per sample and every sample. Prepare fresh daily as needed. 
Combine (20μL) of BME with (880μL) ST1B.
Amount900 µL
For samples preserved in RNAlater
For samples preserved in RNAlater
Remove RNAlater from Sterivex. If clay was used to plug the outlet, unplug with a sterile needle. 
Connect a sterile syringe to the inlet and flush Sterivex with ~ 50mL RNase free H2O. 
* Make sure all water is pushed out before moving forward.
Note
Be sure to Re-plug or Re-cap the outlet.
Alternative for RNAlater removal if pushing liquid through the Sterivex is not an option
Alternative for RNAlater removal if pushing liquid through the Sterivex is not an option
Pull back the plunger of a 3mL syringe to fill the barrel with 1mL (or more) air.
Attach the syringe to the inlet end of the Sterivex and hold the unit vertically with the syringe at the bottom to allow as much of the lysate as possible to be near the inlet. 
Push air into the unit until there is a resistance and release the plunger. You may not feel resistance but if you push and pull air in and out a couple of times, it should work to pull the lysate into the syringe. 
DO NOT FORCE AIR THROUGH THE STERIVEX.
Collect the RNAlater in a centrifuge tube if cells detach from the filter. 
Centrifuge to pellet cells and discard the supernatant. 
Resuspend cells in the 900μL ST1B/BME solution, and add back into the Sterivex through the inlet. Insert pipette completely into the inlet so that the pipette tip is visible inside the unit just above the membrane.  
Note
ST1B/BME solution listed above.
If RNAlater was not used.
If RNAlater was not used.
Remove inlet cap and add (900μL) of the ST1B/BME solution use a pipette tip. Insert pipette completely into the inlet so that the pipette tip is visible inside the unit just above the membrane.  
Amount900 µL
Continue as follows regardless of RNAlater utilization.
Continue as follows regardless of RNAlater utilization.
Recap the inlet and secure the Sterivex filter unit horizontally with the inlet facing out.
Vortex at minimum speed for 5 minutes.
Duration00:05:00
While still attached to the vortex adapter, rotate the Sterivex 180 degrees from the original position. 
Note
Marking initial position on the inlet cap may be useful.
Vortex at minimum speed for an additional 5 minutes. 
Duration00:05:00
Note
An increase in time may be necessary if many cells are still attached to the filter.
Set the Sterivex with the inlet facing up and remove the inlet cap.
Add (900μL) of ST2 solution using a pipette tip and recap the inlet. 
Amount900 µL
Note
Recall ST2 solution must be warmed.
Incubate the Sterivex at 70ºC for 10 minutes, be sure to place unit for equal distribution of heat. 
Duration00:01:00
Cool the unit at room temperature for two minutes. Ensure caps are on tightly, as they may loosen after heating. 
Note
It is importatnt to cool the unit before re-tightening the caps to minimize warping of the inlet.
Secure the Sterivex on the vortex with the inlet facing out n. Vortex at maximum speed for 7.5-10 minutes. 
Note
Critical step for removing the remaining cells from filter and effectively lysing the cells.
Using the provided 3mL syringe, pull back the plunger with 1mL (or more) of air. Attach the syringe to the inlet of the Sterivex and hold the unit vertically with the syringe at the bottom to allow as much of the lysate as possible to be near the inlet. 
Push air into the unit until there is a resistance and release the plunger. Repeat air resistance process until you are able to pull the lysate out into the syringe.  *DO NOT FORCE AIR THROUGH THE STERIVEX*
Add the lysate to the 5mL PowerWater Sterivex glass Bead Tube.


Secure the BeadTube with the lysate to the vortex.
Vortex at maximum speed for 7.5-10 minutes. 
Note
This step is critical for homogenization and lysis of the cells.
Centrifuge the tube at 3600 x g for 2 minutes at room temperature. 
Duration00:02:00
Placing the pipette tip down into the beads against the bottom of the tube, transfer all the supernatant out to a clean 2.2mL collection tube provided in kit. Pipet more than once to ensure removal of supernatant. 
Any carry over of beads will not affect the subsequent steps. 
Note
Expect to recover about 1.5mL of supernatant.
Add 300μL of ST3 solution and vortex briefly to mix
.

Amount300 µL
Incubate at 4ºC for 5 minutes.
Duration00:05:00
Centrifuge the tubes at 13000 x g for 1 minute.
Duration00:01:00
Avoiding the pellet, transfer the supernatant to a clean 5mL collection tube.
Add 1.5mL of 100% RNase-free EtOH and 1.5mL of warmed ST4 Solution, then mix.
Place a binding column (provided) into a 2.2mL microcentrifuge tube (provided). Add previous solution (step 32) to the binding column (800μL) at a time. Centrifuge at 13000 x g for 1 minute and discard the supernatant each time to collect the RNA in the filter.
Note
This step replaces the need to use the vacuum manifold. The vacuum manifold tends to get clogged up when the samples are salty (i.e. with use of RNAlater) and in general takes a long time to actually filter.
When all of the solution from step 32 has been filtered through the binding column completely, centrifuge one final time for 1 minute to ensure the filter is completely dry.
Duration00:01:00
Transfer the binding column to a new 2.2mL microcentrifuge tube.
Add 800 μL of Solution ST5 to the binding column, and let sit for 1 minute. 
Amount800 µL
Centrifuge for 1 minute at 13000 x g.
Duration00:01:00
 Discard the supernatant and centrifuge again for 1 minute to ensure filter is completely dry. Discard residual supernatant. 
Duration00:01:00
Shake solution ST6 to mix well. Add 800 μL of ST6 and let sit for 1 minute. 
Amount800 µL
Duration00:01:00
Centrifuge ST6 solution for 1 minute at 13000 x g. 
Duration00:01:00
Discard the supernatant, and centrifuge again for 1 minute to ensure filter is completely dry. Discard residual supernatant. 
Duration00:01:00
Again, add 800 μL of Solution ST5 to binding column. Let sit for 1 minute. 
Amount1 mL
Duration00:01:00
Centrifuge solution ST5 for 1 minute at 13000 x g.
Discard the supernatant and centrifuge again for 1 minute to ensure the filter is completely dry. Discard the residual supernatant.
If RNAlater was used.
If RNAlater was used.
Add 800μL of 100% RNase free EtOH to the binding column. Let sit for 1 minute.
Centrifuge 100% EtOH for 1 minute at 13000 x g.
Discard the supernatant.
Continue as follows regardless of RNAlater utilization.
Continue as follows regardless of RNAlater utilization.
Centrifuge again for 2 minutes to ensure filter is completely dry. Discard residual supernatant. 
Place binding column into a new 1.5 mL centrifuge tube.
Add 100 μL of RNase-free water to the center of the filter. Let sit for 1-5 minutes.
Amount100 µL
Centrifuge at room temperature for 1 minute at 13,000 x g.
Duration00:01:00
RNA is collected this time, discard the binding column. 
Use Turbo DNase kit for DNase treatment. Follow manufacturer's protocol. 
http:/http://tools.thermofisher.com/content/sfs/manuals/cms_055740.pdf
RNA ready to use, or store at -80º C.