Are you simply trying to get rid of floating culture cells? Simple filtration should do the trick - choose the right pore size depending on your particles of interest.

If you are trying to concentrate viral particles without ultracentrifugation, I recently saw a brochure for a Clontech kit (Lenti-X Concentrator) that allows you to concentrate lentivirus in proprietary tubes without ultracentrifugation. Probably some sort of PEG. I have not tried it myself, but worth checking out.

Dear

Thanks for reply. I have virus grown in cell culture, did centrifuge, took supernatant for virus RNA extraction. However, sequencing facility said that it contain far more cellular RNA than viral RNA. This is my problem

I used Amicon ultra columns Millipore. Now they have something new: Millipore’s Fast-Trap Adenovirus, Lentivirus and Adeno-associated Virus Purification and Concentration Kits provide a fast, safe, and easy alternative for viral purification.

Since viruses tend to protect their RNA in enclosed capsids/nucleocapsids why not simply treat your cell culture supernatants with RNases to get rid of all the floating RNAs and then proceed to extract the RNA using Trizol LS/ phenol chloroform. Theoretically most of the free-floating cellular RNAs should be degraded while your viral RNA would be protected. I normally spin down the sups and filter (0.45um) them to remove as much cell debris as possible, I would at least do that if it's free-floating RNA you're worried about.

Hi Muhammad,

The advice of everyone is great. You will want to use at least 0.45 um filters to first exclude any cells and then I would recommend the Lenti-X kit from Clontech to concentrate the virus. This should get rid of unwanted RNAs while concentrating those you want without the need of an ultracentrifuge. Best of luck,

Daniel

You could try precipitating the virus with ammonium sulphate or polyethylene glycol. This requires relatively low speed centrifugation. You could also try a 300 mw cutoff filter and was with PBS. Any free host RNA will pass through the filter. the virus will be retained.

I'd second what some of the others have said - having tried several of them. to get rid of cellular RNA from lysed cells you can indeed treat the supernatent with RNAse (or DNAse as there will be DNA in it too) and the viral RNA (usually) remains protected in it's capsid, then inactivate the RNase/DNAse and proceed with viral extraction as per normal. To concentrate the virus out of solution without access to a high speed centrifuge PEG precipitation works ok, though you will need to get rid of your cellular RNA first as the PEG will precipitate free nucleic acids as well as your virus capsids. Of course the simplest method to concentrate your nucleic acid is just to heat evaporate some of the liquid (eg in a vacum concentrator or even just on a heat block). Depending on what you want to do with your viral RNA you can also cope with a bit of cellular RNA in there - you can remove that bio-informatically (ie filter the host data out with a computerafterwards)

Amicon Ultra is fine for most situations.

Somehow, you can brief centrifugation your crude soup at 16000 rpm for 2 mins.

Cell debris will be at the bottom and just transfer the soup to amicon and just follow the protocol.

What's your virus?

Dear

It is Paramyxovirus, Newcastel disease virus

Dears

Many thanks for your valuable comments. I believe i can do something to get rid of cellular RNA

I deal with RNA viruses, we usually Freeze Thaw Freeze thaw (cycles) for cellular disruption and then do the simple centrifugation then collect the supernatant and then do the filtration 

Regards

dear Muhammad,

 There are readily  commercial virus RNA isolation kits are available in the market with out going for virus purification. you can use these kits to isolate total RNA of virus and do the PCR confirmation.

I hope I answer the question.

all the best

Yes, freeze thawing and centrifugation will reduce the cellular condamination. Additionally filtering of supernatant with syringe filter of virus can reduce further. For NGS, RNA/DNA extraction kits are available to avoid Cellular DNA, that all are mostly working by filtration methods. All the best.