3 replies to this topic

[ican]

Hello,

I have a problem with DNA purity. This has happened 3 times: I do RNA isolation with the Qiagen RNeasy kit and get a 260/280 ratio of around 2.10 and a 260/230 ratio of around 2.15. RNA concentration is about 1200 ng/ul. So then, I use AMV reverse transcriptase from promega to transcribe the RNA to cDNA. But the ratio of 260/280 I get is around 1.55 and the 260/230 ratio is around 1.80. The concentration is around 1500ng/ul.

As far as the 260/280 ratio is concerned, this doesn't make sense to me! Why would the ratio drop when I make cDNA from RNA?

Another thing I'd like to ask is about the concentration. Assuming I have an x concentration of RNA, would I expect the cDNA concentration to be higher or not?

Thanks!

[Dr Teeth]

ican, on Aug 19 2009, 06:47 AM, said:

Hello,

I have a problem with DNA purity. This has happened 3 times: I do RNA isolation with the Qiagen RNeasy kit and get a 260/280 ratio of around 2.10 and a 260/230 ratio of around 2.15. RNA concentration is about 1200 ng/ul. So then, I use AMV reverse transcriptase from promega to transcribe the RNA to cDNA. But the ratio of 260/280 I get is around 1.55 and the 260/230 ratio is around 1.80. The concentration is around 1500ng/ul.

As far as the 260/280 ratio is concerned, this doesn't make sense to me! Why would the ratio drop when I make cDNA from RNA?

Another thing I'd like to ask is about the concentration. Assuming I have an x concentration of RNA, would I expect the cDNA concentration to be higher or not?

Thanks!

What is your RNA pellet resuspended in vs the cDNA reaction, which is probably mostly water?  Don't forget that 260/280 ratios are greatly impacted by pH.  Thus, a tris-buffered EDTA solution of RNA (likely pH of 8.0) may have a ratio of 2.1, while the same pellet resuspended in water would yield only a 1.5 ratio.  The two are not directly comparable.
Also, I don't see the point in measuring the 260/280 for your cDNA or attempting to quantify it.  Starting with pure RNA and using equal amounts of RNA for your cDNA synthesis should be sufficient.

[TicTacToe]

As far as i know, measuring the OD of cDNA would be meaningless because in the process of cDNA synthesis, too many "junk" (ie. enzyme, EDTA, dNTP, random hexamer/oligomer etc.) were added and they do remain in the cDNA mixture.  So, the OD may be skewed by all these other ingredients.

[Rsm]

Hi,
Sorry I don't know the exact reason, but a drop in the 260/280 ratio seems to be normal...
"A ratio of ~1.8 is generally accepted as“pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA."
(Quote from here)
Cheers,
Minna