RT qPCR and standard curve analsysis

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Loneranger204's picture
RT qPCR and standard curve analsysis

Hi there,

I will be doing some RT qPCR experiments. I need some guidance on this topic as I am new to qPCR:

a) Suppose I am putting 500 ng of input RNA in a reverse transcription mix of 20 ul, What is the concentration of cDNA that I will be getting?

b) How much of cDNA should be put in one well of qPCR plate. Is there a specific range?

c) How to make the standard curve to examine the efficiency of primers and PCR reaction. I have read in the net that a standard curve is generated by plotting Ct values against log of imput. I am not sure how to generate the log of input. In some examples they have used log total RNA (ng) and in some they have used log dilution. Which is the best method and how to generate it?