Hi,

The standard is 1/10 volume of NaAc and 2.5x volume of absolute Ethanol; therefor for a 100ul RNA sample add 10ul of NaAc and 250ul Ethanol. Vortex lightly then put this in the -20 freezer overnight or a -80 Freezer for an hour before spinning down. This should be followed with a 70% ethanol wash to remove excess salts.  The 2.5x volume is the Minimum you can go higher, and up to 4x volumes is sometimes recommended for small RNA molecules such as MiRNA.

If you are expecting very small amounts of RNA then a carrier such as GlycoBlue will help precipitation and also help visualise the RNA pellet. The carrier needs to be added to the solution and mixed before adding the Ethanol though.

Matt

 Here's my rough guide to RNA precipitation (attached): you might want to take a sample you're not too worried about (like an easily prepared bulk control or similar) and run it through several different precipitation methods to see which works best for you.

LiCl has been frequently used to precipitate RNA contaminated by phenols and polysaccharides . LiCl precipitation offers major advantages over other RNA precipitation methods in that it does not efficiently precipitate DNA, protein or carbohydrate (Barlow et al., 1963). http://www.lifetechnologies.com/ru/ru/home/references/ambion-tech-support/rna-isolation/general-articles/the-use-of-licl-precipitation-for-rna-purification.html

Hi Nesreen,

Which extraction protocol are you using? Is it phenol-based? If you provide some info I'll give you more tailored advice.

In short, I suggest two options: if you have problems with polysaccharides, a CTAB-based protocol in the presence of high salt concentration will likely solve the issue. If phenolic compounds are there too, some PVP (polyvinylpyrrolidone) will prevent oxidation to ketones and associated problems.

Alternatively, if you want to get rid of them during the precipitation, instead of sodium acetate/ethanol in the standard amounts, I would use a combination of that and a high salt solution (sodium citrate + NaCl).

As I said, if you post your protocol I'll give you a detailed optimisation.

Cheers,

Ruben

Hello,

Thinking of it, I optimised the extraction protocol for the desert plant Rhazya stricta (http://www.biomedcentral.com/1471-2229/14/2/), and the main problem I found was a devilish RNAse activity, so the CTAB extraction (even with B-mercaptoethanol) or simple Qiagen RNA-easy (also with B-mercatoethanol) yielded mostly degraded RNA. However, a trizol extraction optimised for high carbohydrate content plus a silica column clean up did the trick.

For your reference, I attach the annotated protocol mentioned above. It includes preparation of home-made column cleanups, in case you are short on budget for Qiagen's. If you do not have trizol, I could guess that acid phenol would do as well (as long as you add it directly to the sample, not incubating in a buffer and then adding the phenol), but I cannot promise with the Rhazya harsh RNAse-rich extracts.

Cheers,

Ruben

Sodium acetate 3M yuo can try 8M lithium chloride after prcipitation with Sodium acetate and Iso-propanol. Include PVP 2% in extraction buffer.

Hi Ruben, this method is good when you have a lot of polysaccharides in grain or leaves, and you can get clean RNA.

https://sites.google.com/site/fpistonprotocols/sarcosil

Cheers

Carmen

Hi Ruben, thank you for your valuable notes and remarks. The kit we are using is RNeasy. Unfortunately, it's not possible to switch to trizol or CTAB based protocols.

I think we might  give the second option you proposed a try; using a combination of sodium citrate + and high concentration of NaCl, hopefully we'll result with a higher RNA quality.

Attached is the protocol of the kit we are using(RNeasy). a simple modification was done of adding a re-precipitation step of extracted RNA.

Thank you all for your collaboration, really valuable notes are being posted.

:) Bless You All.

Well, i ve been doing RNA isolation for the hardy plant as well and felt that following a conventional protocol is quite challenging. However, if your major aim is not actually dependent on quantity of RNA, you can opt, using a plant RNA isolation kit. This would give you the quantity of RNA sufficient for RT-PCR.

Hi,

Yes, if it is not possible to switch to buffer-based protocols I would do the precipitation of the column eluate in high-salt buffer.

To try to increase the yield, I'd suggest a try: do the buffer part of the RNAeasy with twice or three times as much the recommended amount of tissue, and then precipitate with the high-salt buffer. Then resuspend in water and do a clean-up through a column. Since you have get rid of polysaccharides before the column, you might increase yield here. Give a go to the home-made Qiagen version I cited (http://www.ncbi.nlm.nih.gov/pubmed/22260178) to make the buffers for clean-up.

My main concern would be the quality of the RNA. If you don't see degradation by RNAses on a gel, you'll be fine... otherwise, I'd strongly suggest trying acid phenol or trizol.

Cheers and good luck,

Ruben

Use 10% volume of 5 M Lithium Chloride and store the sample for 2 hr at -80C