Icycler iq apparatus

Performance and optimal annealing temperatures of the PCR primers for uPA and BRMS1 were tested with gradient PCR. An initial DNA denaturation step at 95° C for 5 min was followed by 35 cycles of denaturation at 95° C for 30 s, primer annealing and extension at 50° C to 70° C for 20 s. The primers for uPA are forward 5′-GGAGATGAAGTTTGAGGTGG-3′ and reverse 5′-GGTCTGTATAGTCCGGGATG-3′ and for BRMS1 are forward 5′‘CTGCCGCC CAGCAAGA-3′ and reverse 5′-GCCTTTTTGATGGCTGTCCA-3′. The primer specificity was confirmed by sequence searches against human DNA databases and analyzed electrophoretically on agarose gels. qPCR was performed using an iCycler iQ apparatus (Bio-Rad) associated with the iCycler Optical System Interface software (version 2.3; Bio-Rad). All PCR analyses were performed in triplicate in a volume of 20 μl, using 96-well optical-grade PCR plates and optical sealing tape (Bio-Rad). Differences in the expression of the uPA and BRMS1 transcripts were normalized with respect to GAPDH expression. The thermal cycling conditions used an initial DNA denaturation step at 95° C for 8 min followed by 35 cycles of denaturation at 95° C for 15 s, annealing and extension at 60° C for 1 min. The relative level of expression was calculated with the formula 2−Δct.