Are you using phenol for the extraction of the RNA? If so this suggests that there is some phenol that is still left over. I would recommend doing a phenol choloform based purification afterwards.

If you are using a nanodrop to detect the RNA concentration, it should tell you what contaminant it is (i.e. phenol, protein, etc.) with an adjusted concentration. If it says it is phenol I always use the adjusted concentration and still go on with cDNA synthesis which is usually fine, but if it is protein, I would not suggest using the samples.

Laurence Stuart Dawkins-Hall

Joshua Beytebiere I am using trizol, I already performed the purification step

Just to follow up, I proceeded with cDNA synthesis and rt-pcr along with samples which had 260/280 and 260/230 ratios of 2s and 1.8/1.9s and the results are pretty much the same in PCR when viewing house keeping gene expression for house keeping genes(ACTB, GAPDH, 18s PPIA and B2M)

Both the comments of Laurence Stuart Dawkins-Hall

Muhammad Uzair Rehman Yes then it sounds like there is an issue with the purification not removing all of the trizol if you have a 260/280 of 1.4-1.5 based on my experience this will cause issues. I would do another round of purification on those samples and that should clear up the issue.

I can provide a paper with a great protocol that I actually use that has an extra extraction step and extra washes that I think (in my experience) help with the purity and yield, please be sure to site the reference if you use this protocol. Please see the pub attached.

Skylar King thank you I will this a try.

Maybe it is not the same case but I have synthesized cDNA from RNA with the following values:

9.4 ng/µL; 260/280 2.31; 260/230 0.54

...and it became into cDNA with these values:

320.7 ng/µL; 260/280 1.81; 260/230 1.05

... and the qPCR worked perfectly.

I hope this answer helps you and more people in this community.