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PROBLEM WITH MELTING CURVES - i realy need help (Mar/03/2010 )

Hello, I hope you can help me, I started a few months ago to use qPCR to quantify my genes and I realize that I understand less and less of this technique.

The most irritating problem that I encountered is that the technical duplicates give me different melting curves .. made with the same mix and the same cDNA, only pipetting twice. It seens that for some reactions it forms primer dimers and for other it din't.

Another thing is that i don't know how alike must be the melting curves to say that is the same product. For example for one of my genes, my samples have peaks at 74 C and other in 74.5 .. is the same?

If the samples have a CT higher than 30 should discard that data? or up to 35 ok?.

Could I perform comparative measurements is I have primer dimers? as I have primer dimers in all my samples..

Extra information:
My genes have lots of homologous sequences because I work with a very repetitive genome.. Anyway I check my primers using blast..
The cycling is

Hold: 95C x 10 min
40 cycles of
95C 15''
60C 40''
Melt: 65 to 94 rysing by 1degree each step,

I'm using Syber Green from Invitrogen, and I check my primers using conventional PCR, using the same cycling.

Thanks..

Anap

-Anap-

Hi,

I wouldn't care about 0.5C difference in melting temperature. That should be the same product. I use melting curves to check if I have one or two peaks (i.e. one or two products). If there's one sharp peak at around the same value than positive control, that's a good reaction.
I recommend running a gel after every qPCR. Mix your duplicates and check size of product. You can also see if there's primer dimer.
I also recommend using a negative control for every qPCR reaction. You will sometimes find a signal from those wells, this signal will tell you where your data starts to be unreliable. So if the Ct of your gene is 38, but your negative control is really negative, or at 45, it might be ok. But if your negative control is at 40 or even 35, you'd better forget about that.
One more, I'm not sure about only 40 seconds at 60C in your protocol. AFAIK, 60s are recommended to complete measurements. But if it works for you... I also usually do 45 cycles. But that's up to you.

Best,

Minna

-Minna-

Minna on Mar 4 2010, 01:56 AM said:

Hi,

I wouldn't care about 0.5C difference in melting temperature. That should be the same product. I use melting curves to check if I have one or two peaks (i.e. one or two products). If there's one sharp peak at around the same value than positive control, that's a good reaction.
I recommend running a gel after every qPCR. Mix your duplicates and check size of product. You can also see if there's primer dimer.
I also recommend using a negative control for every qPCR reaction. You will sometimes find a signal from those wells, this signal will tell you where your data starts to be unreliable. So if the Ct of your gene is 38, but your negative control is really negative, or at 45, it might be ok. But if your negative control is at 40 or even 35, you'd better forget about that.
One more, I'm not sure about only 40 seconds at 60C in your protocol. AFAIK, 60s are recommended to complete measurements. But if it works for you... I also usually do 45 cycles. But that's up to you.

Best,

Minna



Hello, thank u very much for your reply, the thing of the temperature differences was very useful. I'm currently running a gel with the products of the latter reaction.

Anyway, in some cases I have obtained different curves for a reply, it is as if in a tube had primer dimers and not in others, it obviously affects the efficiency of the reaction, but I not always obtain different CT for that reactions. I attached few images, the curves in each graph correspond to repetitions of the same sample.

Respecting the protocol, I use it because it was used in the paper that validated the constitutive primers I am using, I tested it with my primers and it worked, so I didn't change it.
Attached File

-Anap-