The transgene copy number was evaluated using real time quantitative PCR (qPCR) by determining the threshold cycle (Ct) of the transgene and a standard curve generated from a serial dilution of known quantities of the pBI-ΔGR-TetO-EGFP cDNA plasmid [25].
Sample quantitation was performed using a standard curve established from a serial dilution of a mix of the samples.
In fact, a difference of 11,6× between the hTERT levels in plasma from P. vivax patients (1.278 pg/ml) and healthy donors (0.1098 pg/ml) was confirmed when a standard curve, built from a serial dilution of an amplified sample of hTERT sequence, was used to interpolate the hTERT concentrations in the samples (Figure S1).
The standard curve was produced from a serial dilution of a pool consisting of all cDNA samples.
Optical densities (OD) from CCD exposure to Western blot chemiluminescence from known numbers of cyclin B1-expressing cells and from a serial dilution of purified recombinant cyclin B1 were used to determine the average molecules of cyclin B1 per cell.
The relative expressions were calculated based on the efficiency corrected Ct method; the PCR efficiency was estimated from a serial dilution of cDNA generated from pooled RNA of the samples.
After a further treatment at 75 °C for 20 min, the released viral DNA was measured by real-time PCR analysis, using a standard curve derived from a serial dilutions of Namalwa cell lysate, which contain two copies of integrated EBV genome per Namalwa cell.
Relative efficiency was determined using a standard curve generated from a serial dilution of total cDNA (derived from cell lines), which was also used as an internal calibrator between plates.
In brief, real-time PCR reactions of HPV16 E7 were normalised with β-actin and compared with a standard curve generated from a serial dilution of the HPV16-positive cell line SiHa (ATCC-LGC-HTB-35, Manassas, VA, USA) (Schache et al, 2011).
The Northern blot results were quantified using signals obtained from a serial dilution of equivalent in vitro transcripts, which were hybridized using the appropriate probes in parallel.
