TITLE: RNA extraction from field-collected brain tissue samples from suspect rabid animals
AUTHORS: Kirstyn Brunker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [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)
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.
?. [Sample preparation]
Samples stored in RNA later/glycerol-salinePrepare a homogeniser tube by adding 1.4mm ceramic beads (use a 0.2ml PCR tube to measure approx. amount of beads) to a  reinforced tube and then add  ~ of RNA/DNA shield using a pipette or disposable plastic pastetteRemove 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  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 shieldAdd 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 shieldHomogenise the sample for  and then in  (if required) until the sample is fully homogenised. Leave for  to allow sample inactivation.Transfer  of homogenised sample to a new 2ml screw cap tubeAdd  of RNA Lysis Buffer (1:1) and mix well.
2 ml
1 ml
*If the sample has liquefied:Transfer    of liquid to a new 2ml screw cap tube using a pipette or disposable plastic pastetteAdd   of RNase-free water or PBS to the sample (1:1). Then add 4 volumes of RNA Lysis Buffer (4:1) and mix.
[approx.]
[pulses]
Notes on homogenisation:Tissue samples harden in RNA later, therefore may require a longer homogenisationIf the Terralyzer gets hot, leave to cool for few minutes before using againIt 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
350 µl
350 µl
?. [Sample preparation]
Homogenised samples stored in DNA/RNA shieldTransfer  of homogenised sample to a new  screw cap tube using a pipette or disposable plastic pastetteAdd  of RNA Lysis Buffer (1:1) and mix well
350 µl
2 ml
350 µl
?. [RNA extraction]
RNA extraction and purification is performed using the Zymo Research Quick-RNA miniprep kit. The following steps summarise the manufacturer's instructions:
All centrifugation steps should be performed at   -   for    unless otherwise specified.
?. [RNA extraction]
Transfer the sample lysed in RNA Lysis Buffer () into a Spin-Away Filter column (yellow) in a collection tube and centrifuge to remove the majority of genomic DNA. Save the flow-through.
700 µl
To process samples >700 μl, Zymo-Spin columns may be reloaded
?. [RNA extraction]
Add a 1:1 volume of ethanol (95-100%) to the sample flow-through and mix well by pipetting up and down
?. [RNA extraction]
Transfer the mixture to a Zymo-Spin IIICG column (green) in a collection tube and centrifuge. Discard the flow-through.
?. [RNA extraction]
Perform an on-column DNase I treatment: Add  RNA Wash Buffer to the column and centrifuge. Discard the flow-through. In an RNase-free tube, add  DNase I to  DNA Digestion Buffer* and mix. Add the mix directly to the column matrix (try not to touch the filter matrix with the pipette tip).Incubate the column at room temperature for
Prior to use, reconstitute the lyophilized DNase I as indicated on the vial. Store frozen aliquots.
400 µl
5 µl
75 µl
*If preparing multiple samples make a mastermix
?. [RNA extraction]
Add  RNA Prep Buffer to the column and centrifuge. Discard the flow- through.
400 µl
?. [RNA extraction]
Add  RNA Wash Buffer to the column and centrifuge. Discard the flow-through.
700 µl
?. [RNA extraction]
Add  RNA Wash Buffer and centrifuge the column for  to ensure complete removal of the wash buffer. Transfer the column carefully into a eppendorf tube (you can discard the collection tube).
400 µl
1.5 ml
?. [RNA extraction]
Add  DNase/RNase-Free Water directly to the column matrix and centrifuge. Keep the flow-through: this is the purified RNA!
50 µl
The eluted RNA can be used immediately or stored at ≤ .