Protocol Online logo
Top : Forum Archives: : Real-Time PCR

Problems with dissociation curves. - I know this is not new...but its still confusing (Feb/19/2007 )

Hello everyone,
I come to you with a different set of problems. This time I have attached pictures for you veiwing pleasure and also help the problem solving better.

Here is the problem.
I have a primer that I have been using forever for my gene of interest. It produces a 88bp product and is built across exons. However my untreated samples give me twin peaks in this and the CT levels are very low ( around 35-consistently over 3 repetitions) (Fig -Dissn 1) and my treated samples however give single peak (Dissn 1A), since I was not happy with the results, I redesigned the primer to give a bigger product (140 bp) and I get 1 consistent peak in both treated and untreated samples (DIssn 2 and 3), however this is where the problem begins, the fold changes are all completely out of whack for the new primer.
With the old primer, I find that at a certain time point the fold change of treated over untreated are 17 fold (3 different repeats of both RT and PCR), but when I use the new primer, the fold change is 50 fold, which I can accept coz the trend is the same. But when I use an intervention group, the reuslts are conpletely topsy turvy. i.e the samples that showed down regulation are now showing upregulation (by about 500 fold...). SO I am confused.
The most important thing is I have my thesis defense in April and this data has to go in to the thesis. This is a centarl part of my tehsis and I would be skewered alive if the results hold true...please help!!!!
FOr some reason I am not able to upload any of my files.
So if anybody wants to look at the curves, please IM me and will send them the files.
THank you for any and all of your input.
Its highly appreciated

-Brown girl in the ring-

Hi,

could you just describe what you mean with intervention group? At present I don't understand the problem...

-kr├╝melmonster-

Untreated- No treatment of any kind, low exppression of GOI
Treated- Treated with Estrogen, increased expression
Intervention- Prevention agent +E2 - modulated expression

-Brown girl in the ring-