Hi Jaya:

Just from the analysis of your amplification plots, first is not a good idea get amplicons before cycle 10, in some bibliography is considered the lag phase, so probably your cDNA is too much concentrated, so if you obtain 100 microg/microL please test a  mix of all your cDNA samples 1/5 or 1/10 dilutions to obtain a good dynamic range and avoid distortion during qPCR. Second, Is possible to observe in your amplification plots differences in CT value that possible correlate with your experiments, remember that you must collect Ct VALUES ONLY AT THE TRESHOLD LINE, doesn't matter that the curves merge at the end of PCR reaction during saturation phase.  

kind regards

Carlos

Both Dr.Gaete-Eastman and Chinmaya have given very good advice. If I might ask a couple other questions. How have you determined that there was no difference between the knockdown samples versus the controls? How many and what reference genes are you using? If it is only beta-actin, I am sorry to say that it is not the most reliable reference gene on its own (even though publications persist in using it, tubulin, GAPDH, Ubiquitin and ef1a without proper validation). Adenosine diphosphate ribosylation factor (adp-rf, arf in notation) genes work better for normalization, but all reference genes need robust validation with each experiment regardless (sorry if I am just repeating things you already know). From your chart it is difficult to say whether any sample had knockdown unless if the one sample that is amplifying at about 19.5 Ct is your target and not beta-actin. Still I see a good spread of Ct values in your target amplification area, enough to imply some difference between samples. Unfortunately I see a wider distribution in your beta actin amplification plots, which will definitely lead to difficult normalization. Follow the advice above and try to at least get all threshold Ct values to above 20 (although values can still be good between 17 and 20, but again you have to prove that they are accurate, and scale with dilution series predictably). Don't throw in the towel just yet, go through the complete process of normalization first, and then you can make a better guess at whether your goal was achieved or not.

Hello Jaya,

Dr Carlos Gaete-Eastman, Chinmaya Sadangi & Jordan Tan Pepper already have given very good suggestions. I also think that your cDNA may be too much concentrated. If I were you, firstly I will dilute my cDNA . Secondly I will run QPCR to get standard curve to check the efficiency of my primers (range of Efficiency 1.9 to 2.1). In this step, we can optimize the annealing temperature & amount of primers etc. to get optimal efficiency.

Best regards,

Thu