Before you proceed with cDNA preparation from RNA, make sure that you quantify the RNA properly. Quality of RNA should be checked aleast by running it on a gel, If possible go for RIN analysis. After cDNA preparation, run a PCR with internal controls and check it in the gel to see if the concentrations are the same.Nomalise the cDNA concentration for all samples and proceed with real time PCR.
I think you should think of many points:
1-the concentration or the RNA which you use to make your cDNA. i prefer to use nanodrop to adjust the concentration.
2-making the standard plasmid.
Don't use Hosekeeping genes like many researchers do. You should test all your genes you are interested in and then use the 2 or 3 genes that have the lowest differences in your treatment settings. They should be used as your "reference gene". See also MIQE guidelines: (see attachment)
Cheers, Nadine
I have used a software to design my primers. so it is very important if you will design yours you should take care about the false amplicon.
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