I have extracted viral and exosome RNA from culture supernantants. These two sources of RNA are less prone to degradation as they are protected either by the virus capsid or the exosome membrane. I have had good success with the QiAmp Viral RNA kit. This kit uses a lysis buffer (to lyse either the virus or the exosome) followed by binding of the RNA to a column.

maybe if you want to isolate from injured cells, the ARN is degraded if the cell content is in the medium. trizol is used to lyse cells. I think that you can recolect the supernatants and clean it first by centrifugation, and then you can try precipitate the nucleic acid in it. using isopropanol is most used, or maybe, you can combine precipitacion and column-based method. you can check you protocol, to avoid ARN degradation.

Isolation of RNA from supernatant is going to be tricky as RNA degrades fast once it is out of the cell. The first factor to take into account would be the timing of your isolation. You will need to find a perfect time after your cell injury to get the RNA before it degrades. Also, I would think column based method would work best here as it is more sensitive. Specifically, I remember using a kit from Stratagene that was designed to isolate RNA from 100 cells or less. That is the type of kit you will need to be using. As mentioned below, is it viral RNA you are trying to extract? If so, then you will probably need to get a specific Viral RNA kit.

I have extracted viral and exosome RNA from culture supernantants. These two sources of RNA are less prone to degradation as they are protected either by the virus capsid or the exosome membrane. I have had good success with the QiAmp Viral RNA kit. This kit uses a lysis buffer (to lyse either the virus or the exosome) followed by binding of the RNA to a column.

I am trying to extract RNA from the supernatants of human cell cultures. Certainly, RNA extraction is going to be tricky. I will take in account your recommendation, the timing among the injury and the RNA extraction. I will check in detail those kits designed to isolate RNA from "nano" samples. Thanks!

In my opinion, Trizol still yields the best recovery over column RNA isolation. I take 100 uL of supernatant (in my case, freshly-gathered BAL fluid) per 1 mL of Trizol, store at -80C for up to a week, then proceed with typical extraction: thaw, vortex for 5 seconds, add 200 uL nuclease-free chloroform, vortex briefly, let sit 3 minutes, microfuge 15 min at 12,000g (@4C), remove 550 uL of top water layer into a new nuclease-free 1.5 mL tube, and to that, add 500 uL (-20C pre-cooled) nuclease-free isopropanol, vortex briefly, let stand 10 minutes, microfuge 15 min at 12,000g (@4C), pour off isopropanol (carefully, as pellets are not visible, but ARE at the bottom of the tube), add 1 mL (-20C pre-cooled) 70% nuclease-free ethanol, vortex briefly, microfuge 15 minutes at 16300g (@4C), carefully pour off ethanol, let tubes air-dry under fume hood 35 minutes, add 50 uL nuclease-free water back to pellet (still not visible at all, but it IS there), vortex thoroughly (~30 seconds), spin down briefly, heat tubes at 58C for 2 minutes, vortex 5 seconds, spin down briefly, add 29 uL nuclease-free water and 1uL RNAseOUT (to 0.5 U/uL), then store until use at 4C (to avoid freeze-thaw; relying on RNAseOUT to do its work). Works every time (for qPCR application). It is the proportion of supernatant per volume of Trizol that is key, in addition to recovering at least 550 uL of the top water layer during the extraction process. You lose about 12.7% of the water layer (that you want to leave behind anyway to avoid the protein/gDNA interface while gathering the water layer).

The other key (as others have mentioned above, is to gather the supernatant as soon as you can (when it is "ready") to avoid certain degradation of the sought-after RNA by [already] cell-released supernatant RNAses. In saying that... you may be actually be facing an impossibility. I.e., perhaps your RNA in most cases IS already degraded before you can get it into the Trizol (or like reagent) in the first place. So your situation could already be a futile game of cat chases tail if this be the case. E.g., lysed macrophages notoriously release a lot of RNases. Perhaps add RNAseOUT to your supernatants as early on as you can to thwart RNase degradation as early as possible. But RNAseOUT is expensive.

I have done that before. However it happens right after I lyse the cell. As we know, RNase, which can damage RNA, naturally present in the environment and even in cells, so if you want to extract the RNA it will largely depent on timing and terperature issue. Maybe you can apply some RNase inhibitor to avoid the damage.

Might I recommend the paper www.ncbi.nlm.nih.gov/pubmed/22424315 where they compare various approaches.

As you proposed, I will need to add RNase inhibitors in the supernatants.

I will check this interesting paper!

Thanks!

(I would like to reanimate this topic, if possible). I am going to do some RNA extraction from conditioned medium and wondering if it is obligatory to use so big volumes of TRIzol for extraction. I incubate cells in serum-free medium, i.e. there should be quite small amount of protein which theoretically may reduce amount of TRIzol required for efficient lysis. @Jack M. Gallup, or anybody else - did you try other proportions of TRIzol:medium volumes? Or it should be strictly 10:1 in any case? (yes I know about TRIzol LS, I just don't have it available at this moment)

Similarly, I am wondering if I can use just acidic phenol for first steps. I know that for cells/tissues/etc TRIzol is more convenient but maybe in case of protein-free medium it will give no special advantage?

I am confused that what is the difference between extraction RNA from the medium and from the exosome? 

why we isolate RNA instead of DNA during cell culture