Is it possible to add RNALater directly to bacterial cultures instead of centrifuging first and then resuspending them in RNALater? If yes, then what should be the ratio of cell suspension:RNALater?
Hello Péter,
I don't know this but I thing this is no problem. The centrifugation is only to take away the media. But when you see this: http://www.ebiotrade.com/buyf/productsf/qiagen/76506.htm you can see that they mix before centrifugation.
I think you may should the bacteria sediment and then take away most of the media and then mix with RNALater.
To the best of my knowledge (and quickly checking the RNAlater online protocol) they don't offer a means to directly add the solution to your bacterial culture.
I've previously worked with RNAprotect from Qiagen, and this solution can be added to your bacterial cultures at a ratio of 5 parts RNAprotect : 1 part media. They recommend not exceeding a total volume of 1mL or a bacterial number greater than 1 x 10^7. In my own experience, we've stabilized RNA in 5x10^7 - 1x10^8 bacterial cells (bifidobacteria) without any problems (resuspending pellets in 1ml of 100% RNAprotect). Once the bacteria have been suspended in the solution, allow them to sit for 10 minutes prior to putting in the -80 if you're storing them, in order to allow for cell penetration.
Best of luck!
Indeed, RNAprotect is the best for bacterial cultures but I have a lot of RNALater and I was just curious whether I could use it simply by mixing. Obviously I cannot. I think I will take Dr. Dittmann's advice and after centrifuging and a quick resuspension in the remaining media I will mix the cell suspension with 10 volume of RNALater.
Thank you for your quick response!
The question is whether you want to use RNALater to preserve the RNA or to freeze reactions to study subtle gene expression changes that took place beforehand. Depending on the strain that you use 5 minutes may be enough to gross changes in the expression pattern. Therefore in these cases centrifugation is simply not an option. We usually freeze biochemical reactions (inactivate all enzymes) by mixing the culture with equal volume of ice-cold Ehtanol, containing 5% Phenol. THEN you can harvest the cells, apply RNALater and store the samples on RT or send by mail or do whatever.
I know that Phenol-EtOH is just the opposite philosophy compared to RNALater, though.
Interesting. I have never heard of this ethanol/phenol combo before. I will certainly try it next time which will be soon since the current sampling has just turned out to be too early according to the analytics. Thank you for this valuable information!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA