Briefly, the DNA was mixed with the primer pool, containing all primers for generating the 224 amplicons and the AmpliSeq HiFi Master Mix, and transferred to a PCR cycler (BioRad, Munich, Germany). PCR cycling conditions were as follows: Initial denaturation: 99°C for 2 min, cycling: ...
Real-time-PCR was performed in a reaction volume of 20 μl containing 2 μl cDNA, Light Cycler TaqMan Master (Roche), primers and probes for p21, EGFR and the housekeeping gene porphobilinogen deaminase (PBGD) in concentrations recommended by the manufacturer (Real Time Ready Assays, Roche...
0.5 to 2 μg mRNA was reverse transcribed in a reaction mix containing 10U/μL Moloney Murine Leukemia Virus reverse transcriptase (Life Technologies), 5 ng/ μL Random Primers (Life Technologies), 1x first strand buffer (Life Technologies), 5 mM DTT (Life Technologies),...