Hi,
I am having major problems in isolating RNA from activated (and not) RAW cells. Apparently it looks like they undergo very rapid degradation. I isolated with TRIZOL with the standard protocol as I am always doing for microglial cells (macrophage-like) and never had this problem. I am looking for microRNA with TAQMAN and in some samples even the endogenous control is at 30ct in the qPCR. On top of that it's really difficult to see low expressed genes. For sybergreen I actually see multiple melt peaks with oligos that are usually perfect! So I am suspecting the RNA is degradated..(?)
Does anyone has experienced that too? Any clue or help?
Thanks!
Flavia
The best, cheapest way to check for RNA degradation is to just run a small portion on an agarose gel. You should see sharp ribosomal RNA bands, smearing or a poor ratio of large:small ribosomal bands indicates RNA has degraded. Best of luck!
Did you use a spectrophotometer(e.g. Nanodrop or nanoquant) to assess the quality of RNA. The integrity of RNA however can be checked by running a 1% denaturing AGE. Please clarify what do you mean by activated cells? I mean how are they treated? and what are low expressed genes? (Do you mean miR genes?)Ct at 30 is possible depending upon transcript's copy number, unless the isolated fraction of RNA is good. Multiple melt peaks indicate non-specificity. Please check the primers once again. If they are Ok, it means that the RNA quality was compromised.
Hi,
I am going to check the integrity with Bioanalyzer soon enough. The cells were treated with LPS 10ng/ml but I can see some problem as well in untreated cells. I checked for miR but sometimes I have u6 at 30, or at 24, or 26 when in most of the samples is about 18 (so only some samples looks bad, but they are too much to believe my data in general).
The oligos are always fine in other cell type and unfortunately I am studying a very difficult gene rich of GC and repeated sequences. Anyway didn't have that many peaks in my melt, the maximum was in really complex samples in which I had a larger one (most probably one attached to another).
Has the toxicity been checked? It might be deleterious to gene expression, else as you said RNA is guilty as cliched. Make sure that its RIN>=7(Bioanalyzer). Rectify using standardization as per MIQE guidelines. You wont ever face any problems again.
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