2 replies to this topic

[rtpcrnovice]

Hello All,

As name suggests, I'm new to PCR world. I have extracted total RNA and our measure of 260/280 are mostly around 2.0 (1.9-2.1) which i realize is good. However, our 260/230 ratios vary anywhere between 1.5 to 2.0. Reading through various forums, I realize that >1.8 is desirable. My question though is "IS IT WORTH THE TIME/RESOURCES TO AIM FOR 1.8?' Will i run later into any questionable data if I don't get above/close to 1.8?

In other words, can I consider 1.5 close to 1.8 and proceed or should I target for 1.8?

I use Trizol-Chloroform extraction method, and nondrop for obtaining  260/280 and 260/230 ratios.

Many thanks.
G.

[ivanbio]

If you have 260/280 ratios around 2.0 then I would not worry about 260/230 ratios as low as 1.5. While it is nice to have very clean RNA (260/230 > 1.8), the reality is that some tissues/samples never give you squeaky clean RNA.

Shoot for 260/280 ratios around 2.0 and make sure to always run internal controls when you analyze the expression of your genes.

[rtpcrnovice]

ivanbio, on Mar 6 2010, 06:59 PM, said:

If you have 260/280 ratios around 2.0 then I would not worry about 260/230 ratios as low as 1.5. While it is nice to have very clean RNA (260/230 > 1.8), the reality is that some tissues/samples never give you squeaky clean RNA.

Shoot for 260/280 ratios around 2.0 and make sure to always run internal controls when you analyze the expression of your genes.

Thanks Ivan. That was helpful.
As long as I have an internal control (e.g. actin) quantified in the same sample, I should be OK. The assumption being that whatever effect of phenol and other contaminants on reverse transcriptase, it would be reflected on both gene of interest and internal control.
Many thanks,
-G