Iam using Trizol for RNA expression experiment
Here is the concentration and Purity
Any recommendations for enhancing the concentration and purity
How to eliminate phenolic contamination that give absorbancevat 260-270 nm also
Hey Fatma. try the following modifications: ensure that there is no precipitate and complete Trizol lysis of ur sample. elute with 25-30 microliter. do not skip DNAse treatment and let it for 15 mins. work as quick as you can. try to reduce your trizol to sample ration:for example use 500 microliter of trizol instead of 1000 microliter. do not overheat your sample and try to work on ice. repeat the washing procedure for 3 times instead of twice. hope to find your answer here.
Raw values are less useful than the actual traces: we can't see the 260/230 ratio, for example (which is usually more important than 260/280), and nor can we necessarily confirm that the low 260/280 is due to phenol at 270 (rather than protein at 280).
Also, what is your starting material (cells? Tissue?) and how much are you using (and how much TRIzol)?
Ok, so first: it's serum. Cell free biofluid, and thus minimally transcriptionally active. There will not be a lot of RNA, total, in your sample (and most of it will be miRs).
I would not, honestly, expect to get anything quantifiable from 100ul of serum, regardless of method. You might get a reading via qbit/ribogreen approaches, but nanodrop? No.
You could simply assume you have some RNA, and push forward to cDNA synthesis and see if you can see anything (that's what I usually do for serum), but again, it depends what you're looking for. If you're looking for miRs, then this should work. If you're looking for mRNA, on the other hand, then...you might not see much.
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