Impure RNA - problem with cDNA? (Sep/19/2005 )

Hi Gents and Ladies

I was wondering during this never ending night if

-- the RNA i 've just extracted (500 ng/microliter) is ok for cDNA sinteshis....the 260/280 is 1.58....i want to do cDNA against another set of RNA (1.78).....

--I want to use the same concentration...do cDNA and then a PCR, just to see on a gel if there's some gross differences (before do a Real Time ......better know if the gene is expressed or not?)

Someone think is a problem having two different purity level?

Thanks

Luca,

Hi, different levels of purity cant really be balanced, if you try to make them match you will dilute one sample. It is total RNA you have, i take it is the mRNA you are interested in? You know how much total RNA you have but you dont know how much of it is mRNA therefore i dont think you can balance it out for qRT PCR. If you are looking at gene expression then if something is upregulated you should still spot it, working on the basis that a cell has 1 copy of a particular gene in one situ and 2 copies in another situ then in theory you should have a distinguishable amount after PCR as the one band on a gel would be twice as bright as the other and quantifying it would give you the same data

I have found that if you start with RNA at purities less than 1.7-1.8, you will get funny results when you get to real-time PCR

by funny, I mean your data will be inconsistent. You can certainly try it, but your replications may not match and if, at a later date, you repeat the experiment with cleaner RNA you may not see the same data trend.

been there, been burned...good luck

Thanks a lot, now i'm just looking at RT PCR level to see presence absence....

Then , when i will swithc to real time i will keep in mind the purity problem!!


Luca,