Research Article

Effects of Palm Vitamin E on Bone-Formation-Related Gene Expression in Nicotine-Treated Rats

Table 2

SYBR Green real-time PCR reaction mixture and protocol.

Reaction mixture
Master mixVolume per one reaction

2X SYBR Green RT-PCR reaction mix12.5  𝜇 L
Forward primer (10  𝜇 M)1  𝜇 L
Reverse primer (10  𝜇 M)1  𝜇 L
Nuclease-free H2O9  𝜇 L
RNA template (1 pg to 100 ng total RNA)1  𝜇 L
iScript reverse transcriptase for one-step RT-PCR0.5  𝜇 L
Final reactions volume25  𝜇 L


cDNA synthesis20 min at 50°C
iScript reverse transcriptase inactivation4 min at 95°C
PCR cycling and detection
(standard PCR cycle)
Denaturation 10 sec at 95°C
Annealing/extension30 sec at 61°C (data collection step)
Repeated for (30 to 45 cycles)
1 min at 95°C
Melt curve analysis (optional)1 min at 55°C
10 sec at 55°C (80 cycles, increasing each by 0.5°C each cycle)