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: ...
and 2μg of total rna. the quality of rna was checked by gapdh pcr and only samples positive for gapdh transcripts were used for analysis. real-time-pcr was performed in a reaction volume of 20μl containing 2μl cdna, light cycler taqman master (roche), primers and probes for p21, ...
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)...