standard curve for AQ analysis

1 post / 0 new
ifhmn
ifhmn's picture
standard curve for AQ analysis

Hi there,
I have synthesized cDNA from 0.3ug of RNA in a total 20uL of reaction. I cannot increase the yield of my cDNA since that I have 2 samples with low concentration of RNA. Than I dilute all of my cDNA 5 times (1:5) and pooled them together where I diluted 8uL of my cDNA in 32uL of RT-PCR water . Then I made a 1:2 of dilution series of my pooled cDNA where I mixed 50uL of cDNA with 50uL of RT-PCR water. As a result, I have 5 dilution points. Then I put 4uL of my diluted cDNA in a total 20uL of qPCR reaction in order to make standard curve for my housekeeping gene and GOI. Can someone tell how calculate copy number of my cDNA in order to design a standard curve?  Below are the results of my qPCR for my standard:
A) Houskeeping gene (UBQ10)               B)GOI
Standard   Ct                                         Standard    Ct
A               16.498                                       A            24.996 
B               17.598                                       B             28.963
C               18.791                                       C            32.398
D               19.523                                       D            33.779
E               20.381                                        E             36.03

Is my cDNA to diluted? should I repeat again my qPCR?
My samples are from plant material