2 replies to this topic

[krisw]

Hi there,

What are your guidelines for omitting points on a standard curve? I am running gene expression assays with Taqman on ABI 7500. Some of my runs are perfect, others not so much. In one particular case, my R2 is a bit too low, 0.977. If I omit the very lowest point in the standard curve (all three replicates) all the values (R2, slope) improve. My samples fall closer to the other end of the standard curve so there is not problem there. But I feel that I am cheating a little bit, because there is nothing actually wrong with that low point. It doesn't get flagged by the software, all the replicates are nice and tight. How do you feel about this practice?

On the same topic, what about eliminating one replicate of a standard curve point that is visually off from the other two but is not so far off that the software flags it?

Thanks for any help. We just got this instrument into our lab and will be using it a lot. I want to establish solid analysis guidelines to follow.

Kris

[tea-test]

If dilution points of your std curve are outside the dynamic range of your assay you HAVE TO exclude them or they will screw up your results.
You are also not allowed to quantify samples that are lying outside your std curve.

what dilution range are you using for your standard curve? with high ranges of dilution (>5logs) r2 values of >0.99 are easily achieved.

[krisw]

Thanks good to hear. Yes, I follow ABI instructions and have std curve ranging from 0.4ng to 250ng. Of course, sometimes I adjust these up or down so the samples will fall within the range.