Samples were run using the Lightcycler programme, with a 15-minute incubation at 95°C followed by 45 thermal cycles, consisting of a 15-second denaturation step at 94°C, then a 20-second annealing step at 60°C, and a 20-second extension step at 72°C after which fluorescence was read.
This included a 30 seconds denaturation step at 95°C, a 30 seconds annealing step at 55°C (Figα) or 57°C (PAR6 and β-actin), and a 40 seconds extension step at 72°C.
The initial PCR activation step was at 94°C for four minutes, followed by the denaturation step at 94°C for one minute, primer-annealing step at 55°C for 30 seconds, extension step at 72°C for one minute, and the final extension step at 72°C for ten minutes.
PCR was performed for 40 cycles comprising a 60-second denaturation step at 94°C, a 60-second annealing step at 55°C, a 90-second extension step at 72°C followed by 10 minutes at 72°C.
PCR was performed at 55 degrees Celsius, 32 amplification cycles, 60 seconds extension step.
The Tc_CLB1 amplicon of 3 kb size uses a 45 seconds extension step, in comparison with the other amplicons that uses just 10 seconds.
8) The second primer-extension reaction using the fluorescently labelled 3′- O- 2-cyanoethyl -dNTP derivatives 2– 5 and the reverse transcriptase was carried O- 2-cyanoethyl -dNTPn cO- 2-cyanoethyl -dNTPO- 2-cyanoethyl -dNTPn step (step 2).
After 6-minute initial denaturation at 96°C, a "hot start" amplification was initiated by adding DreamTaq DNA polymerase, followed by 19 cycles consisting of a 15 seconds denaturation step at 95°C, a 30 seconds annealing step at 64°C, and a 15 seconds extension step at 72°C, with a final extension for 45 minutes at 72°C.
Quantitative PCR was performed with the following programme: pre-incubation at 50°C for 2 minutes, initial denaturation at 95°C for 5 minutes, 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, elongation at 72°C for 30 seconds, and a 20-second final extension step.
Reactions for qPCR were prepared using the Corbett robotics machine (Qiagen, UK) and performed on the MJ Research Chromo 4TM (Genetic Research Instrumentation Ltd ,Essex, UK) using the following program: 95°c for 5 minutes, followed by 42 cycles of a 30 second 95°C denaturation, 30 second 61°C annealing and 30 second 72°C extension steps.
Real-time PCR reactions were performed at 95°C for 15 minutes, followed by 40 cycles of amplification consisting of denaturation step at 95°C for 15 seconds and extension step at 60°C for 1 minute.
