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The first change – the removal of the quantification threshold – could have a major effect on federal agency practice.
From our data, we estimated a quantification threshold for temperature variation of 1.2 °C.
CEQ removed the quantification threshold and now recommends that agencies quantify direct and indirect GHG emissions whenever the tools, methodologies, and data are available to do so.
Furthermore, amplification efficiency monotonically decreases as DNA concentration increases, in contrast with the assumption that amplification efficiency is constant below the quantification threshold.
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For quantification, thresholds were set so that the subset of cells with strong nuclear accumulation were scored as positive.
To determine primer efficiencies and optimal quantification thresholds, a dilution series of a cDNA mix consisting of 4 ul solution from every sample was created.
All quantifications (threshold cycle [ C T] values) were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed to determine the relative level of gene expression.
Relative quantity (RQ) for each sample was calculated using the formula, 1/ECq, where Cq is the quantification cycle (threshold cycle).
PCR efficiencies and optimal quantification cycle threshold (Cq values were estimated using the online Real time PCR Miner tool [ 29].
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