I'm doing a gene expression analysis in peanut roots. I was working with actin as housekeeping gene, and all was working good till one day... the melt curve and Ct values didn't work anymore. I'm changing different parameters in my reaction… like using a different master mix, other oligos, water, etc... and the curve and ct values are still bad! Somebody can help me?
The file attached contains results from the same samples.
Except for the weaker peak from the data points represented by the open purple boxes, I don't see a problem with your data. What exactly do you mean by "bad"? Besides, the melt curve is the least informative and least important part of a qPCR experiment. If your primers have acceptable efficiency and your controls all worked, then you have interpretable data.
I believe that you are worried about the non-specific amplification. Do you reconfirm the pcr product by gel electrophoresis? If you get two different bands, there could be some other primer contamination which is a common handling error while working with multiple genes.
hi. Dr. Johan Rodríguez
you don't get a good melt curve in my housekeeping gene , this may be due to the primer or milt temp.
i suggest you read this
Interpreting melt curves: An indicator, not a diagnosis
http://www.science.smith.edu/cmbs/wp-content/uploads/sites/36/2015/09/Interpreting-melt-curves.pdf
Melting Curve Analysis
https://www.fluidigm.com/binaries/content/documents/fluidigm/resources/melting-curve-analysis-ug-68000118/melting-curve-analysis-ug-68000118/fluidigm%3Afile
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