For each run, serially diluted cDNA of K562 cells was used as a quantitative standard.
Serial dilutions of in vitro transcribed RNA generated from a linearized plasmid encoding nucleotides 136 400 of the 5' UTR of GBV-C with GenBank accession number U44402[ 14] was used as a quantitative standard curve.
Hemagglutinin gene fragments, amplified with primers designed according to the published sequence of the H7 subtype (GenBank No. KC853228), were inserted into the recombinant T plasmids (TaKaRa) and were used as a quantitative standard.
Invasive coronary angiography (ICA) served as the standard of reference, using a quantitative approach.
Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp) is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard.
Based on converting target quantity from fluorescence units (F0) to the number of target molecules (N0), the central premise of optical calibration is that absolute scale can be established using a single quantitative standard, if it can be assumed that all amplicons generate similar fluorescence intensities.
HCV RNA titer is calculated using a competitive quantitative standard, obviating the need for the laboratory to perform calibrations.
In either case, it was evident that the ease of conducting absolute qPCR would be greatly increased by implementing optical calibration using a universal quantitative standard.
This contention has been corroborated through performance testing using lambda gDNA as a quantitative standard, which has generated many datasets illustrating the exceptional quantitative capabilities of real-time qPCR (e.g. Figures 5, 6 and 7; also see [7]; data not shown).
The serum and SF FSTL1 concentrations were detected using a standard quantitative sandwich ELISA (Groundwork Biotechnology Diagnosticate, San Diego, CA, US) with a detection limit of 100 pg/ml.
