DIY RNA Spin Column Buffers – Purification of RNA with humble DNA silica spin columns

Published: March 30th, 2020   Last Modified: April 20th, 2020

Personally I don’t have much experience with RNA spin columns. We’re more of a Trizol/RNAzol lab ourselves (DIY recipe for Trizol, thanks Julian, wherever you are). However the convenience and speed of spin columns cannot be denied. Unlike DNA silica purification, there is less known about brewing your own buffers.

I’ve put off reverse engineering these recipes, but I think it’s finally time. This page is a work in progress, I’ll update it when I have data over the next little while. Readers are encouraged to try the buffers themselves and leave a comment, or get in touch so I can put up your data (With full credit, of course).

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!!! Don’t want to hear me rambling? GIMME DA RECIPES !!!

Currently one of the most popular RNA extraction kits is the Qiagen RNeasy kit. Unfortunately, unlike their DNA extraction buffers, the recipes are not easily obtainable. I doubt that they’ll be releasing their recipes anytime soon, but who knows, we may be pleasantly surprised!

Until then, let’s see what we can do! From early trawling you can find a patent by Qiagen where they attempt to replace ethanol in their buffers with less hazardous solvents for the purposes of shipping. I don’t think they ever sold a product with this technology, but it does reveal a few putative recipes. If we assume that they used their glycol replacements 1:1 with ethanol:

Buffer RW1 (RNA Wash 1?)
20% Ethanol
900 mM GITC
10 mM Tris-HCl pH 7.5

Buffer RPE (RNA Pre-Elution?)
80% Ethanol
100 mM NaCl
10 mM Tris-HCl pH 7.5

Elution Buffer
Nuclease free water, (“Ladies and gentlemen, we got em.”)

So, it’s something, but we’re missing the critical component of the kit, Buffer RLT. This buffer is used for lysis and cell disruption, and allows the RNA to bind to the silica column. What do we know about Buffer RLT? It has guanidium iso/thiocyanite (GITC), and it needs to be diluted 1:1 with 70% ethanol for binding to occur. We can infer that they likely used the same pH and buffer. Also, the SDS states that the GITC concentration is between 30-50% by weight, which equals to 2.5M to 4.2M. As well, the pH of the mixture is 7.0

Putative Buffer RLT
2.5-4.2M of GITC
10 mM Tris-HCl pH 7.0
Other components?

Okay, okay, not bad, not bad. We’re in the right ballpark of GITC concentrations when we think of DNA binding. Once diluted 1:1, 1.25M seems a bit low for nucleic acid binding. Most final concentrations of GITC we’ve seen previously are in the 1.6-2.2M range. But we’re still not sure, or at least not sure enough to continue…are we?

A patent from Agilent comes to the rescue here, where they focus primarily on making improvements of the membrane in a spin column, using different materials etc. Here are their recipes with some of the legalese removed and the ingredient concentrations inferred from the patent:

Lysis Buffer
4M GITC
143mM β-ME (Add before using, don’t store large amounts)
25mM Tris-HCl, pH 7

Stabilization Solution (For cleanup of previously isolated/synthesized RNA)
4M GITC
25mM Tris, pH 7

Wash Buffer #1:
1M GITC,
25 mM Tris-HCl pH 7
10% ethanol

Wash Buffer #2:
25 mM Tris-HCl pH 7
70% EtOH

Wash Buffer #3 (For use with Stabilizing solution):
25mM Tris-HCl pH 7,
70% Ethanol

Ohh, we have you now! Lets line up the recipes side by side. Added recipes from Roche High Pure RNA Isolation Kit, thank you kind reader (Alex) and thank you Roche for not making me dig through patents. Added recipes from papers suggested on twitter. Keep it coming 🙂

Powerpoint file for easy editing

So, what do you think? Looking pretty similar side by side. Agilent tests their buffers on their improved membranes AND silica membranes, which Qiagen uses. This means that the above recipes can be used on columns meant for DNA isolation/purification/minipreps etc. This is confirmed in the following paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282632/

Considering Agilent also dilutes their lysis buffer 1:1 with 70% ethanol before binding onto the column, here’s my best guess at the recipe of Buffer RLT:

Buffer RLT, putative but likely
4M GITC
25mM Tris-HCl pH 7.0

Whew! Now to actually test these buffers, results coming soon, readers who do it quicker can get in touch and I can put up your results.

If you don’t have GITC available, guanidine hydrochloride (GuHCl) can be substituted. If you don’t have that, sodium iodide (NaI) can be substituted. If you don’t have that either….well, I’m open to suggestions, urea maybe? Note, substituted chemicals will need tweaking of concentrations.

Additional Useful Reading on RNA Extraction Protocols:

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203011

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282632/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503482/

https://bomb.bio/wp-content/uploads/2018/09/8.2_BOMB_total_RNA_extraction_mammalian_GITC_V1.0.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2245989/#!po=40.1316