Yes. As you want to quantify the gene expression, you need to design the primers should fall in cds region, generally.  Before proceed to experiment, set a semi-quantitative PCR to ensure single band amplification.

Thanks brother. If the both said properties are present in primers, lies in cds region and showing single band  on GEL, then what could be the cause of dimers &  peaks below threshold line in qPCR ?

Hi Hassan,

Your primers being CDS-specific and giving a single band on gel do not guarantee that you won't get primer dimers. It is possible that primers dimers are too small to be visible on your gel. What percent gel did you use? I would recommend taking a closer look on your primers to check whether or not they can form dimers. If they do, design a new set of primers or play with the concentration of your current primers and use concentration that does not result in dimer formation. Secondly, how does your melting curve look? Do you see two peaks?

Also check out this link:

https://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2014/01/20/interpreting-melt-curves-an-indicator-not-a-diagnosis

Good luck,

Hi

basically primers for RT-PCR should amplify a 50-200 base pairs region of your gene of interest and they must have same T of annealing. I also highly recommend you to design the forward at the end of one exon and the reverse at the beginning of the next exon (exon spanning primers), in this way you will not unspecifically target genomic DNA

Hope it helps  

Dear Mohsan and Andrea, really thanks for your valuable comments. I had used 0.7% Agarose GEL and the band was quite good as i checked it with gradient PCR, it gave good band at temperatures ranging from 56 to 60 so i used 58 for qPCR. Kindly check the attached pics for peaks i got from qPCR for one target gene in different plant samples.

The gel and melt curves support each other. There appears to be non-specific product accumulation in the higher Tms. Can you confirm the bright band you are seeing is the predicted amplicon size? 

I agree with Can. There seem to be primer dimers in your conventional PCR reactions. I would suggest playing around with the primer concentrations. Usually, we tend to add primers in a huge abundance to our PCR reactions, and their concentrations can be safely decreased without affecting the amplification of specific amplicon.

Dear Mohsan & Can Thanks for your comments. GEL is showing the bands of same PCR product at different temperatures, performed just to check opt temperature for qPCR. Primer amplicon size was 111 and i thought it was good. what you people say about this ?

That's good, but do you remember/record the predicted amplicon size when you did the primer design?

If it's near ~110 then you can say that amplicon (from gel) is the target of interest, not product of non-specific amplication.

Please also analyze this figure as this is the same primer i used with different tissues of plant. Here it gives relatively good result in 2nd and 3rd tissue samples. The first one that i shared was another tissue i had done with same primers.

I would exclude any replicate from analysis that has a curve between 75-84*C melt curves as your main amplicon Tm seems to be around 85.

It is so kind of you that you are guiding me in this very situation. So i can consider those curves which do not lie between 75-84 C but they are below threshold line ? If yes what it shows ? either the expression is very low or there is any type of contamination / mishandling or anything else ?