Hy everybody, I am looking for a way to stop gene expression during harvesting of microalgae to ensure reliable RNAseq in response to temperature. I am affraid working on ice and performing centrifugation at 4°C and bieng quick is not enough...
I saw that RNA protect for cell i(qiagen) costs about 300 € for 250 mL and that 5 Volume have to be added to the culture media.... For me each sample volume is 50mL so this is NOT affordable. Any other clue how I could easily prevent gene expression from the time I start collecting the cells? Many thanks
I do not know whether this is feasible, but one approach would be to centrifuge cells at the same temperature they had been exposed to, instead of low temperature.
Hi,
Would a fixation with 4% PFA or equivalent be feasible? It is a strategy to reduce mortality in FACS and people seem to report good quality RNA extracted from PFA fixated cells.
In addition to Daniel Pouly suggestion about PFA, since you are doing RNASeq you are primarily concerned with RNA and not with changes in protein (correct?). In which case you could just add a transcriptional inhibitor like Actinomycin D (for eukaryotes).
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