Hi Ranjan,

Cawthon published 2 different protocols, one in `02 and one in `09.  Which version are you using? (the `09 version has a pretty cool primer design for the SCG).  We typically get CT values in the 20 cycles range (with 10 ng loaded, using the 2002 version).

Something worth checking is the primer concentration! (not joking.  last week we were having some efficiency issues with 36B4.  Correcting the primer concentration helped a lot).

Hope this helps.

--Matt

Thanks Matt,

Presently we are doing this assay in mouse NIH3T3 cells using primer combinations mentioned in articles of Calaghan and Fenech, Biol Procedures Online, 13: 3, 2011; Callicott and Womack, Compartive Med, 56: 17, 2006. We will start the assay in human samples soon. I am not sure whether the expression of 36B4 in mouse cells is low or our procedure is not optimized yet? Another problem we are facing is the NTC peak in dissociation curve is overlapping with the product peak. We have tried all the available options. It is coming in both the Telomere and 36B4 PCR. We are doing in separate plate. Do you think changing the melt curve program can improve the NTC results without affecting the product peak? Presently we are using 95oC x 15s, 55oC x 30s, 95oC x 15s with 0.1oC/s.

Thanks again.

Ranjan

the expression pattern shouldn't matte since you should be targeting the gene itself and not it mRNA transcript.  I would check to make sure the primers will amplify mouse DNA. 

For the NTC having some DNA in it that is amplifying, that should only be a real problem if it is crossing threshold at a similar cycle as your target (preferably it is shifted several cycles to the right, the more the better).  Our NTC melting curve has typically shown a slightly lower Tm than target.  If they are the same, your NTC has target DNA in it (36B4 or telomere).  if they aren't the same you are likely seeing either primer dimer formation or some other off-target affect.  If you were to run the products on a gel (2% agarose) the NTC band wouldn't be the same as the target. (as a heads up the telomere bands won't be sharp bands if you do this).

As for changing the melt curve program, that won't effect your data interpretation any, it is only there for QC purposes (other products formed, etc...)

--Matt

Thanks Matt,

I will update you the results in future.

Hi Matt,

The NTC Ct values for both telomere and 36B4 are way behind the (8-10 Ct) the Ct value of the last standard curve points. So in that sense it is ok. But when I see the melt curve, it is coming at the same temp of the product, i.e, 80oC. We have changed the primers (re ordered), water and master mix but not much success.