Hi Soo, what is the concentration of RNA in your samples? Sometimes, very low RNA concentrations result in aberrant 260/280 ratios. Secondly, 260/280 ratio is not reflective of DNA contamination, since both DNA and RNA have maximum absorbance at 260 nm. On another note, do you mix your DNase with RDD (the buffer for DNase) before pouring to the column?

What will you use your RNA for? If this is for qPCR, you should design primers that span exon-exon junctions, and stop worrying about DNA contamination. 

Good luck!

Hello Mohsan Saeed :) thank you for your answer.

My RNA concentration is about 450ng/mul and yes I do mix DNase with RDD befor pouring to the column.

I will use my RNA to make first strand cDNA and try PCR with some of virus primers since I have no idea what this virus is. all I want to know is the sequence of this virus, do you have better options for sequencing? (I want to try old way because NGS seems too expensive for me)

thanks :)

Do you use NanoDrop to measure RNA concentration? How do you blank your instrument? Hope you are using the same buffer to blank the spectrophotometer that you use to elute your RNA. Can you dilute your RNA in water and see if this improves the 260/280 ratio? Although it will not solve the problem, but you will get some sense if there is "something" in your prep that is giving aberrant 260/280 ratio and if you can dilute that "something", and get a better ratio. 

For sequencing, I guess you are on the right (but risky) tract. If you don't want to perform NGS, the best way is to do some intelligent guess work and use a bunch of primers with the hope that you will be able to get amplification.