That's the bit they've not mastered yet, their mixture of interesting textures and pilfered melodies is never quite convincing on its own.
The PCR analysis was performed on cDNA in a master mixture (ReddyMix PCR Master Mix, ABgene House, Blenheim Road, UK) containing 20 pmol of each primer.
Gene expression of FOXA2, C/EBPalpha, CYP51, HSP105B and HNF6 was determined with primers reported in [3] in a standard PCR reaction containing 50 ng of DNA, 4.0 mM MgCl2 and 2 µl of LightCycler DNA Master hybridisation mixture (LightCycler DNA Master Hybridization Probes, Roche Diagnostics Inc) in a total volume of 20 µl.
The amplification mixture consisted of 2 μl of 10× LightCycler FastStart DNA Master Hybridization Probes mixture (Roche), 2 mM MgCl2, 0.5 μM each primer, 0.2 μM each probe, and 5 μl of template DNA in a final volume of 20 μl.
Master mixture contained 0.5 µmol/L primers, 0.4 μmol/L probe, LightCycler TaqMan Reaction Mixture (Roche Applied Sciences), and water.
PCR amplification in a 20 μl reaction volume consisted of a master mixture containing DNA Taq polymerase, dNTP mixture and buffer (LightCycler DNA Master SYBR Green 1, Roche Diagnostics, catalog no. 2158817), 4 mM MgCl2, 0.9 μM of each primer and cDNA in a glass capillary tube.
Master mixture 2 (MM2) shows the positivity of genotypes HPV 18, 39, 45, 59 and 68 and master mixture 3 (MM3) reveals the positivity of HPV genotypes 16, 31, 33, 35, 52 and 58.
For each PCR, a new master mixture was created to mitigate issues with a single batch and to minimize contamination of the master mixture.
A master mixture of QNASBA (Quantitative Nucleic Acid Sequence Based Amplification) was used to quantify marker proteins by real time amplification of the aptamers.
The cDNA was amplified using a SYBR Green master mixture (Bio-Red, USA) with a LightCycler 96 (Roche, USA).
