Dear All,
I am working on quantitative real-time PCR assays in the detection of viruses. In that is it better to have the quantitative standard dilutions of higher concentrations ranges or to have lower concentrations ranges? For ex: 1E+7, 1E+6, 1E+5, 1E+4 IU/ml or 1E+4, 1E+3, 1E+2, 1E+1 IU/ml?
Both? There really is no need to restrict yourself to a four-point curve when you're using a technique that can easily handle 7-8 orders of magnitude.
You need to measure both extremes, really, to determine
1) the upper range (very abundant targets cannot be accurately quantified, so you need to know how high you can get before the assay isn't quantitative)
2) the lower level of detection (can you detect single viruses, and is your assay entirely negative when there are no targets, or do you have a no-target baseline from primer dimers etc)
3) the lower level of quantification (what is the lowest viral titre you can both detect and reliably quantify?) -this may not be the same as 2), above: my assay for viral DNA can detect single viruses, but I would not trust it to be reliably quantitative for numbers below the '20-50 viruses per well' level.
Also, be cautious in your quantification anyway: a value of '13455 viruses' should be reported as "13,000" at best, and probably '~1e4' would be more appropriate.
To minimize the risk of cross-contamination and If the last dilution (1E+1) is higher than the limit detection, you can use five points (e.g. 1E+5, 1E+4, 1E+3, 1E+2, 1E+1).
Aissa Saidi you mean, higher dilutions e.g 1E+6, 1E+7, 1E+8 would cross contaminate the PCR reagents heavily than the lower dilutions?
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