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 mix	Volume per one reaction
2X SYBR Green RT-PCR reaction mix	12.5 𝜇L
Forward primer (10 𝜇M)	1 𝜇L
Reverse primer (10 𝜇M)	1 𝜇L
Nuclease-free H2O	9 𝜇L
RNA template (1 pg to 100 ng total RNA)	1 𝜇L
iScript reverse transcriptase for one-step RT-PCR	0.5 𝜇L
Final reactions volume	25 𝜇L
Protocol
Process	Duration/temperature
cDNA synthesis	20 min at 50°C
iScript reverse transcriptase inactivation	4 min at 95°C
PCR cycling and detection; (standard PCR cycle)
Denaturation	10 sec at 95°C
Annealing/extension	30 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)