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This is due, as discussed in a previous review of influenza-VLP quantification methods [ 42], to the particularity of each quantification method and their intrinsic bias.
For quantification of each gene transcript, an external calibration curve was obtained by plotting the concentration of each quantification calibrator expressed as copies/μL vs corresponding quantification cycle (Cq).
Since ccn1 and ccn2 are members of the same gene family, it is critically important to examine the specificity of each quantification system in order to avoid possible cross-recognition [ 4].
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Further, we utilized these RNA-seq datasets to measure the specificity and sensitivity of each gene quantification method examined in this study.
The ratio of immunopositive nuclei to the number of all cells in each area of quantification and the mean of these ratios for each WSI were sent for computer-enabled statistical analysis.
To simplify, unicate quantifications were applied (only one Cq-measurement of each target per quantification).
Quantification was carried out using a LightCycler 480 Real-Time PCR System (Roche Diagnostics, Mannheim, Germany) by comparing the quantification cycle of each sample to the quantification cycle of a standard curve constructed by amplifying different known copy numbers of target gene.
The current gold standard and method of choice is a time-consuming manual analysis that includes plotting of each vessel and quantification of flow in every individual vessel in a video performed by an experienced user [11].
Initial quality control of each library included quantification of: the titer of each one, the frequency of recombinant/insert-less clones and the average insert size (using a subset of 20 recombinant clones).
However, given there is only one example of each, and no quantification, this statement seems too strong.
The limits of linear quantification of each of the assays were determined with PCR efficiencies of 100±8% and linearities (Pearson's correlation) >0.99.
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