Hi Christina,

I believe the white precipitation you describe will most likely be salt from your RNA later.

RNA in very high concentration can form a cloudy white precipitate when you add ethanol or isopropanol to your aqueous phase from your RNA extraction. However with your case it sounds like salt due to the hard sand like nature of the precipitate.

Can you extract your RNA straight from your cultures rather than using RNA later as I think this would solve your problem.

I have never used RNA later before so there may be a protocol you need to follow when you are ready to extract RNA from your samples.

Hope that is of some help to you,

George

Hi Christina,

I believe the white precipitation you describe will most likely be salt from your RNA later.

RNA in very high concentration can form a cloudy white precipitate when you add ethanol or isopropanol to your aqueous phase from your RNA extraction. However with your case it sounds like salt due to the hard sand like nature of the precipitate.

Can you extract your RNA straight from your cultures rather than using RNA later as I think this would solve your problem.

I have never used RNA later before so there may be a protocol you need to follow when you are ready to extract RNA from your samples.

Hope that is of some help to you,

George

The RiboPure kit is designed to be used with RNAlater, so long as you follow the manufacturer protocols and use the appropriate amount of cells. Using too many cells with this kit will absolutely cause failure.

I suspect that you may have accidentally taken some of the interphase along with the aqueous phase after separation. It's very difficult to see the interphase with this kit in my experience, so I always left behind about 10% of the aqueous volume. The interphase material may come out of solution along with the RNA after the addition of ethanol. I've used this kit with E. coli about a dozen times, but I've never seen the precipitate.

Good luck with your project!

Mark

Hi George. Thank you for your answer.

Salt precipitation might be an explanation... But I remove RNALater by centrifugation before extracting the cells. The precipitate is so big that I can hardly think is coming from RNALater. What do you think?

Mark. The hypothesis of taking along the interface has already came along among the members of my lab, but I ensure I'm very careful and even left a portion of the aqueous phase to avoid taking it. Nevertheless, cell debris have not a "sand or salt like" texture. And as I said, the precipitate was huge. How is possible it came from a so small fraction as interface is?

Could it be any kind of impurity that I've taken all along the process that would precipitate after ethanol addition?

Hi Cristina,

Yes I thought there would be some kind of clean up step to try and remove the RNA later. You said that this has happened only once so is it possible that the clean up step was not as effective for that extraction?

To rule out salt precipitation, you could add some RNA later to water and then add ethanol as you would for the extraction and the salt will precipitate out. Compare the precipitate to the one you observed from your extraction to see if they look similar.

Good luck finding an answer,

George

This sounds like traces of phenol; I have experienced this problem after phenol/chloroform extractions; therefore, as suggested by others, leave more of the aqueous phase behind.

Maybe (but i guess not as you describe it as a hard precipitate) it is the polysaccharides of the LPS layer from gram-... I'm working with plants and when i add the ethanol in a RNA prep kit, polysaccharides precipitate (in my case they are gel like cause they come from mucilage). They cause clogging of tubes when they are transferred

I agree Norbert. Discarded salt hypothesis, polysaccharides are likely to be the problem. We also work with plants and to eliminate sugars we perform 5 minute heating at 65ºC and 15 minute incubation on ice. Then precipitation with ultracentrifuge and you may recover your RNA into new vial. Maybe you can try this. On the other hand I do not know if this could fit in the kit and protocol you are using.

Roger: Good to know i give it a try.. At which point you heat it.. I will do extraction without kit anyway it was just a test pack from a company..

Hi Cristine.

If you isolate RNA with Trisol, it is possible after addition of phenol solution leave the suspension for 10-15 minutes at room temperature, then centrifuge precipitate at 4 C for 10 min at maximum speed, and only then to add chloroform to the supernatant. Some contaminants are not soluble in phenol but soluble in the organic medium after the addition of chloroform, and can thus be removed. Continue to act according to instructions.

We perform phenolic extraction without kit using using guanidine hydrochloride (Logeman 1987 Improved method for the isolation of RNA from plant tissues). I described the very final step of the protocol we use to get rid of sugars. This is the issue why I doubt it could be easy to apply in Cristina's case but let it on the air.

You could be carrying salts from your culturing media. Try to wash your cells with ice cold water before starting phenol extraction.

Originally (historically) nucleic acids were precipitated with Ethanol, and people learned when to use LiCl, Na acetate, ammonium salt or something else.

But by now it has turned out that Ethanol is not needed at all for precipitation RNA. Therefore, beyond the above mentioned factors to be considered, I would recommend you using only LiCl for precipitating RNA. Half molar final concentration will precipitate all RNA on room temperature in a few minutes, higher concentration does not help, lower temperature (ice) may help a bit.

We usually use RNA in applications where DNA would be a problem (like RT-PCR) so we have omitted Ethanol altogether as a main factor that we caused problems for ourselves with.

Of course RNA is not in very high amount in the liquid, so thorough centrifugation is needed afterwards.

We also found abundant white Ethanol-precipitates with plants/Trizol (polisaccharides or polyphenols or whatever in old leaves), so the protocol had to be modified because getting back the RNA from the precipitate is just hopeless. Nevertheless keep in mind that RNA is not stable on high temperature with bivalent cations, so use appropriate amount of EDTA. I myself would consider heating up things even to room temperature as a last chance.

I have the same problem before. 1) Now please make sure your centrifuge work well and able to have maxi-speed to separate the 2 phases. 2) After the centrifugation, do not transfer the aqueous phase to a new tube immediately, but wait for 10-30min.

Totally agree with George Mcilroy. We had the same problem years ago. The white precipitate is due to high salt concentration. Increase wash steps with 70% ethanol. Good Luck

Thank you very much for your comments and recommendations.

The RNA extraction protocol I'm trying has been used in the lab for years, and the person how is training me has never see this kind of precipitate.

I will observe if the same phenomenon happens again. But as I will be in charge to apply it with a bacterial strain that is high producers of a extracellular polysaccharide that may disturb during the process, I will consider some of the recommendations you suggest to improve it, overall those related with polysaccharides precipitation.

Thank you again.

One more thing concerning the storage in RNALater solution. I've read that when working with tissue, it's necessary to keep the sample at +4 overnight, to let the solution penetrate throughout the sample, prior to storage at -80.

Is the +4 step really necessary when working with bacterial cells?

Does any one have experience at this point?

Thank you

could we use Ethanol instead of isopropanol for RNA extraction from human cells?? and tell me did any one use the combination of ethanol and isopropanol for RNA extraction??

If you have phosphate buffer, it precipitates in EtOH and the cold. Try precipitating with isopropanol at room temperature. Add some glycogen if your concentrations are low.