Page banner

#TestingMethods2020

Beads based method for RNA extraction

D1: Path Lab Bulletin
D3: Catalogue of Resources

This is a Binding Buffer that we use to purify RNA:

Reagent

Volume/amount

Final concentration

Guanidine hydrochloride

23.88g

5 M

Water

to 30 ml

 

Isopropanol

to 50 ml

40%

Tween 20

25 µl

0.05%

3M sodium acetate

2 ml

115 mM

Add salt to a falcon tube. Fill up with water to 30 ml. Mix to dissolve the salt (if necessary heat it in the microwave very briefly). Fill up to 50 ml with isopropanol. Add Tween 20 and sodium acetate last.

TET buffer

Reagent

Volume/amount

Final concentration

Water (RNAse free)

~49.4 ml

 

0.5 M EDTA, pH 8.0

100 µl

1 mM

1 M Tris-HCL, pH 8.0

500 µl

10 mM

Tween-20

25 µl

0.05%

Add ~45ml Water in a falcon tube, ad 0.5M EDTA, 1 M Tris-HCL and Tween-20. Fill up to 50ml with Water, swirl very well to ensure Tween-20 is well mixed in. Prepare beads (for 96 samples): After vigorous vortexing of magnetic silica beads, take 550 μL (5 μL bead suspension used per sample, 70 μL extra included) in a 1.5 mL tube. Pellet on magnet and wash twice with 500 μL TET by vortexing and spinning down before pelleting on magnet. Resuspend beads in 1000 μL TET.

Prepare binding buffer/bead mix: In a 50 mL bottle, mix 50ml binding buffer (5M GuHCl, 40% isopropanol, 115 mM NaAc, 0.05% Tween), and the beads. Mix vigorously and pour into a reservoir right before you start the protocol.

For full SOP please see https://www.crick.ac.uk/covid19-consortium

Have you validated this method, if so, how and what were the results of the validation?​

Yes.

By running samples that have been validated previously through an RNA extraction kit.

The purified RNA has been subjected to qPCR. The obtained CT values and curves were found corresponding to the ones obtained with the RNA purified through a kit.

How quickly could this be deployed and what are the dependencies?​

The preparation of the BB takes 20-30 minutes. There are no dependencies as the preparation of the solution requires the use of basic equipment.

We are currently using this approach for testing.

What is the likely production volume?​

From 5-10 litres of the Binding Buffer. We use 50mL in each run.

What are the risks and barriers to using this at scale?​

The solution can be kept at RT for a month

If stored at RT for more than a month it may present precipitates.

Who are you already partnering with on this?

We are performing this in our laboratory pipeline. Full SOPs can be found on

https://www.crick.ac.uk/covid19-consortium

 

 

edited on Apr 10, 2020 by Sonia Gandhi
Commenting is closed

Jo Martin 5 months ago

Good stuff and thanks for sharing!

Sue Hill 5 months ago

This is also being used elsewhere - what special precautions need to be followed to make up this binding buffer ?

Alexander James Phillips 4 months ago

This is being used at the Crick to test NHS samples: https://www.crick.ac.uk/research/covid-19/covid19-consortium

Graham Taylor 5 months ago

Presumably the extraction could be happening during sample transport?

Sam Roberts 5 months ago

Sounds very interesting. Can you tell us a little more about the lab set up required to do this at scale? For example for 1000+ tests a day.

Davide Danovi 5 months ago

Sam and Sonia, we would be really happy to explore joint opportunities to help scaling and Open Cell may be able to help also with establishing an outside sampling station neighbouring. I'd be very happy to give more context.

Rob King 5 months ago

Status label added: D

Kyle Beacham 5 months ago

The idea has been progressed to the next milestone.

Bev Matthews 4 months ago

Status label added: D1

Status label removed: D

Tom Jordan 4 months ago

Thank you for sharing this with us Sonia. We have added this page to our weekly Testing Methods bulletin, to encourage labs to consider this in detail, test and scale up.

You can access the weekly bulletins from the COVID-19 resources hub on the RCPath website: https://www.rcpath.org/profession/coronavirus-resource-hub.html

Kind regards,
Tom Jordan
On behalf of the Testing Methods team

Tom Jordan 4 months ago

The idea has been progressed to the next milestone.

Charlotte Cookson 4 months ago

Hi Sonia, we are trying to make contact to see if you would like to join a conversation we are planning for next week. Charlotte

Users tagged:

Tom Jordan 2 months ago

Status label added: D3: Catalogue of Resources

Share