Mar 07, 2020

Public workspaceRNA extraction from field-collected brain tissue samples from suspect rabid animals

DOI
dx.doi.org/10.17504/protocols.io.bdcei2te
  • 1University of Glasgow
Martha Luka
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bdcei2te
Protocol Citation: Kirstyn Brunker 2020. RNA extraction from field-collected brain tissue samples from suspect rabid animals. protocols.io https://dx.doi.org/10.17504/protocols.io.bdcei2te
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
We use this protocol and it's working
Created: March 06, 2020
Last Modified: October 28, 2024
Protocol Integer ID: 33894
Abstract
This protocol details the steps involved to perform RNA extraction on rabies virus brain tissue samples collected by the WHO recommended "straw method" in the field (Meslin F-X, Kaplan MM, Koprowski H. Laboratory techniques in rabies. World Health Organization; 1996.[10]). The protocol is field-friendly and can be performed using portable, battery powered equipment.
Materials
MATERIALS
ReagentDNA decontaminating solution (DNAaway, 10% bleach, etc.)
Reagent1.5ml Eppendorf tubes
ReagentEthanol
ReagentRNaseZap™ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
ReagentDNA/RNA ShieldZymo ResearchCatalog #R1100-50
ReagentmySPIN™ 12 Mini CentrifugeThermo FisherCatalog #75004081
ReagentTerralyzerZymo ResearchCatalog #S6022
ReagentQuick-RNA Miniprep KitZymo ResearchCatalog #R1054
ReagentWooden applicator stick / toothpicks
ReagentFisherbrand Reinforced 2ml tubes with screw caps a nd o-rings quantity 500 RNase/DNase freeFisher ScientificCatalog #15545809
ReagentCeramic Beads 1.4 mm QiagenCatalog #13113-325
ReagentFilter paper
Safety warnings
Rabies samples must be handled in containment level (CL) 2+ or CL3 conditions until sample inactivation (i.e. section 1 of the protocol). In the field or low resource settings samples can be processed to the point of inactivation in a portable glove box (e.g. UY-33666-50, Cole Parmer). Sample handlers must wear appropriate personal protective equipment, have received training and rabies pre-exposure prophylaxis prior to handling samples.
Before start
Prepare the biosafety cabinet or portable glovebox:
  • Decontaminate surfaces and pipettes with UV (15mins) then wipe down with decontamination wipes or 10% bleach solution and RNAseZap
  • Ensure you have a waste bag, 10% bleach filled waste pot and spray and all consumables/reagents for section 1 inside the glovebox
If samples are frozen, allow to defrost and equilibrate to room temperature
Sample preparation
Sample preparation
Brain tissue samples collected in the field may be stored in glycerol-saline, RNA Later or DNA/RNA shield according to the resources available to the sample collector. Instructions to process commonly received samples for use with the Zymo Research Quick-RNA miniprep kit are indicated below (for other sample types please refer to the kit instruction manual)

Note
DNase I should be included in the kit (R1054/R1055) but please confirm this is the case before beginning - we have experienced that this is not always the case for certain versions of the kit that may still be in distribution.

Homogenised samples stored in DNA/RNA shield
  • Transfer Amount350 µL of homogenised sample to a new Amount2 mL screw cap tube using a pipette or disposable plastic pastette
  • Add Amount350 µL of RNA Lysis Buffer (1:1) and mix well

Samples stored in RNA later/glycerol-saline
  • Prepare a homogeniser tube by adding 1.4mm ceramic beads (use a 0.2ml PCR tube to measure approx. amount of beads) to a Amount2 mL reinforced tube and then add ~Amount1 mL of RNA/DNA shield using a pipette or disposable plastic pastette
  • Remove a small piece of tissue* (50-100mg) from RNA later/glycerol using a wooden applicator stick/toothpick/forceps and dab excess liquid on filter paper
Note
*If the sample has liquefied:
  • Transfer Amount200 µL of liquid to a new 2ml screw cap tube using a pipette or disposable plastic pastette
  • Add Amount200 µL of RNase-free water or PBS to the sample (1:1). Then add 4 volumes of RNA Lysis Buffer (4:1) and mix.

  • Add tissue to the prepared homogeniser tube and ensure the lid is screwed on securely
  • Insert tube into the lysis chamber on the Terralyzer and replace chamber shield
  • Homogenise the sample for Duration00:02:00 approx. and then in Duration00:00:30 pulses (if required) until the sample is fully homogenised.
Note
Notes on homogenisation:
  • Tissue samples harden in RNA later, therefore may require a longer homogenisation
  • If the Terralyzer gets hot, leave to cool for few minutes before using again
  • It may be difficult to see if the sample is fully homogenised due to foam- leave so settle for a few minutes and homogenise again if required

  • Leave for Duration00:02:00 to allow sample inactivation.
  • Transfer Amount350 µL of homogenised sample to a new 2ml screw cap tube
  • Add Amount350 µL of RNA Lysis Buffer (1:1) and mix well.

RNA extraction
RNA extraction
RNA extraction and purification is performed using the Zymo Research Quick-RNA miniprep kit. The following steps summarise the manufacturer's instructions:
Note
All centrifugation steps should be performed at Centrifigation10000 x g - Centrifigation16000 x g for Duration00:00:30 unless otherwise specified.

Transfer the sample lysed in RNA Lysis Buffer (Amount700 µL ) into a Spin-Away Filter column (yellow) in a collection tube and centrifuge to remove the majority of genomic DNA. Save the flow-through.
Note
To process samples >700 μl, Zymo-Spin columns may be reloaded

Add a 1:1 volume of ethanol (95-100%) to the sample flow-through and mix well by pipetting up and down
Transfer the mixture to a Zymo-Spin IIICG column (green) in a collection tube and centrifuge. Discard the flow-through.
Perform an on-column DNase I treatment:
Note
Prior to use, reconstitute the lyophilized DNase I as indicated on the vial. Store frozen aliquots.

  1. Add Amount400 µL RNA Wash Buffer to the column and centrifuge. Discard the flow-through.
  2. In an RNase-free tube, add Amount5 µL DNase I to Amount75 µL DNA Digestion Buffer* and mix. Add the mix directly to the column matrix (try not to touch the filter matrix with the pipette tip).
  3. Incubate the column at room temperature for Duration00:15:00
Note
*If preparing multiple samples make a mastermix

Add Amount400 µL RNA Prep Buffer to the column and centrifuge. Discard the flow- through.
Add Amount700 µL RNA Wash Buffer to the column and centrifuge. Discard the flow-through.

Add Amount400 µL RNA Wash Buffer and centrifuge the column for Duration00:02:00 to ensure complete removal of the wash buffer. Transfer the column carefully into a Amount1.5 mL eppendorf tube (you can discard the collection tube).

Add Amount50 µL DNase/RNase-Free Water directly to the column matrix and centrifuge. Keep the flow-through: this is the purified RNA!

Note
The eluted RNA can be used immediately or stored at ≤Temperature-70 °C .