Hi there,
I am getting different heights for the amplification curves when I set up my standard curve. I usually get the first curve always lower than the latter curves. (graph 1: 98% efficiency, graph 2: 93% graph, 3: 105% efficiency). I have 5 others more that have this trend.
I use 1uL (contains 500ng) of RNA for cDNA synthesis (superscript III from invitrogen) and use 1uL of the cDNA reaction for my first point, and I dilute them 10 fold serially. (my 2nd, 3rd and 4th points). I use Roche's Essential green sybr green mix.
I run it for 10 sec at 95'C, and 45 sec at 58'C.
Is there a possibility that this might affect the efficiency of the PCR reaction?
This is because the high concentrations contain more cDNA. All this cDNA give some signal from SYBR Green, so they have a systematically higher starting values for the fluorescence. This startig value is subtracted from the amplification curves to make the ct values comparable. After some number of cycles, there is so much PCT product in the tubes that all SYBR Green is bound and gives signal. Further amplification does not further increase the signal. This is where the amplification curves reach their plateu. As the total amount of SYBR Green is similar in all tubes, the plateau values differ by the amount of background that was corrected. You can see this when you turn off the background correction (or look at the unprocessed fluorescence measurements) - if the software allows it,
The height difference is probably attributed due to differences in master mix volume between wells i.e more nucleotides for the experiment to go on for longer.
For the most reliable CT values, you want them to be flat at the same time.
This is because the high concentrations contain more cDNA. All this cDNA give some signal from SYBR Green, so they have a systematically higher starting values for the fluorescence. This startig value is subtracted from the amplification curves to make the ct values comparable. After some number of cycles, there is so much PCT product in the tubes that all SYBR Green is bound and gives signal. Further amplification does not further increase the signal. This is where the amplification curves reach their plateu. As the total amount of SYBR Green is similar in all tubes, the plateau values differ by the amount of background that was corrected. You can see this when you turn off the background correction (or look at the unprocessed fluorescence measurements) - if the software allows it,
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