Validating amplification efficiencies of the target and the housekeeping genes - (Dec/19/2006 )
Hi, i am new to RT-PCR and planning to carry out the comparative RT-PCR method. I understand that I need to show that the amplification efficiencies of my target and the housekeeping genes must be approximately equal. What is the best way to go about this? It seems that my software linked to the Lightcycler machine is not able to plot Ct vs template concentration. If I have to plot it manually, and since this is not a linear relationship, how to i go about plotting it? pls advise !!! would be very grateful ....
U can plot d graph using Excel,
plot Ct Values Vs Log Conc (template) ....since d relationship is not linear......for both target gene and housekeeping gene...calculate the slopes. If the slopes for the two graphs match fairly....u can conclude that amplification efficiencies of the two genes are comparable and approx equal.
to get good graph,
prepare std curve using 10-fold diln series....plot Log conc on X-axis and Ct on Y-axis