I have performed qRT-PCR in Bio-Rad CFX96. The melting curve result shows two peaks in some samples within replicates but not all. What might be the cause? And, based on these attachments, would anyone please suggest me how to interpret those results? As, you can see, G1 G2 and G3 are the replicates of single biological sample and N1, N2 and N3 are also the replicates.
Based on your melt curve you shouldn't try to interpret the normalized expression results whatsoever and instead focus on figuring out why you're getting such inconsistent melt curve peaks. There are more than 2 peaks in that melt curve. Even if the "peak" at 80 wouldn't be there, there seem to be multiple peaks between 84 and 88. There are lots of great resources to get you on your way, including one from Bio-Rad, http://www.bio-rad.com/en-ca/applications-technologies/real-time-pcr-troubleshooting
Splt the melt curve diagrams by gene. Otherwise we can't see anything. It would also be helpfult to label the negative and the positive controls.
There Can be several reasons, but pipetting error is the most common reason for this type of scenario.
Most of your primers seem to produce unspecific products (possibly primer-dimers) when the target concentration is low. You may fiddle around with higher annealing temperatures, lower Mg++ concentration or simply try another buffer brand (this sometimes helps!). However, the most promising approach is to design different primers.
I used 60 degree centigrade for annealing and extension (combined) temperature during qPCR because primers melting temperature was around 62 for all forward and reverse primers except actin. Gradient PCR results showed single band in 1.5% gel prior to qPCR.
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