Dear Prof. Stefano Falone

You may use ultrafiltration to remove RNA from conditioned media.

Ultrafiltration works by forcing liquid through a semi-permeable membrane with a specific pore size or molecular weight cut-off (MWCO). Molecules larger than the MWCO will be retained on the filter, while smaller molecules will pass through. To ensure effective RNA removal from conditioned media, the MWCO of the filter should be significantly smaller than the RNA's molecular weight so that smaller molecules like proteins and metabolites can pass through. If the MWCO is close to or larger than the RNA's size, some RNA might pass through.

PES (polyethersulfone) and RC (regenerated cellulose) membranes with MWCO of 50kDa - 100 kDa are commonly used in ultrafiltration to separate intact RNA from conditioned media. These membranes are effective at rejecting most RNA molecules due to their pore size, which is designed to allow smaller molecules to pass through while retaining larger ones, like the intact RNA. Both PES and RC membranes are commonly used in ultrafiltration for their good chemical stability, compatibility with biological samples, and ability to achieve high flux rates.

The specific RCF values used may need to be adjusted based on the specific filter used, the sample volume, and the desired level of purification. For a 50-100 kDa MWCO membrane, to separate RNA from conditioned media, you may use a centrifugation speed ranging between 1,000 to 3,500 x g.

I would suggest not to use the RNase digestion method. Though this method is effective for removing RNA from conditioned media, it may not be suitable in case your goal is to retain other components of the conditioned media.

Regards,

Malcolm Nobre

Dear Colleague,

I will definitely look up for PES-based ultrafiltration methods applicable to my specific case.

Thanks a lot for your prompt and detailed reply and suggestion.

Best ragards

Stefano