I was extracting RNA from honey bee midguts for qPCR using Trizol and my A260/280 values prior to cDNA generation are all around 2.5; my collaborator suspects Trizol contamination. Do you agree and in general, any tips to cleanup the samples?
Some example values:
1) A260: 16.076
A260/A280: 2.41
ng/microliter: 643
2)11.682, 2.45, 467.3 (A260, A260/A280, conc)
3) 9.937, 2.44, 397.5
4) 22.117, 2.41, 884.7
Hi Andrey, I think, your collaborator might be right or did you do a phenol-chloroform precipitation to purify your RNA? In such case, trace amount of phenol may give a higher/abnormal A260/A280 ratio. I trued to make cDNA from RNA sample with range of A260/A280 ratio. Sometimes it worked, sometimes not. My suggestion would be, give a try. Best wishes!
Hey Sharmin, thanks for the reply. I did perform a Chloroform, then Isopropyl, then ethanol wash after adding the trizol. I'll try to update the thread when i get back the results, we don't really have a feasible economic option but to give it a shot. I'm the farthest from an expert but the phenol absorption spectrum looks flatter at 280 to me, so I was surprised to hear it might be Trizol since I left a relatively fair amount above the trizol layer when pipetting.
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