To dear Researchers,
I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8 and it was consistence with that in Scatter plot equation. I also drew a line plot. In the line plot the slope was -3.2 and the efficiency was 100%. Also, there was no linearity in Scatter plot but there was a well fitted linearity (0.99) in line plot. I'm not sure what is the problem. Please help me figure it out.
Kind regards
Alireza
A picture or two would help enormously, as I'm not sure I entirely understand your problem at present. Are you saying you can draw a line through your data if you plot it as a line chart, but not if you use an X-Y scatter plot?
How are you plotting your data? What values are you using, and what are you plotting on each axis?
I mean, a gradient of -3.2 is about what you'd expect if all was well, so all else being equal you're probably fine, but still: more details (or a picture or two) would be useful.
Hi! Yes I think a picture of the two graphs would be really helpful to understand what is going on. Also could you give us a little details on the concentration series you did? Which dilution point did you use and if you can put also the Ct value you observed, we can try to recreate the analysis in Excel to share it with you :)
-3.2 sounds perfect though, usually I use the scatter plot function in Excel, but I have never tried with the line plot.
Dear John Hildyard and Clara Apicella
Thank you so much and I'm sorry for not providing the result at the first place. Actually, the results was for one of the student asking me to see the results. Like always I tried to draw a xy scatter plot and see what is gong on. The slope was bad but when I saw the CTs and concentrations it seemed OK. So I manually evaluated the slope and it was the same as that in the scatter plot. Here, I attached the excel file. It is not actually the exact data since I haven't seen the student yet but they look exactly as I've seen. I personally think this might be as a result of serial concentration. Since values of concentrations (0.1-0.0001) are very far from value 1.
Regards
Ok, had a look.
The problem is the student is plotting Cq (a log2 metric) against concentration (a linear metric), which gives you a ridiculous skew that is impossible to fit a line to. You can fit a log-curve to it (really well, for obvious reasons), but this is not how you do generally do it.
You either linearise your Cq values, or log transform your concentrations (the latter is much easier, and arguably more informative, here).
This (a log/log plot) generates linear data, with (as expected) the gradient of -3.28.
I've attached my edited version.
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