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 mix | Volume per one reaction |
| 2X SYBR Green RT-PCR reaction mix | 12.5 L | Forward primer (10 M) | 1 L | Reverse primer (10 M) | 1 L | Nuclease-free H2O | 9 L | RNA template (1 pg to 100 ng total RNA) | 1 L | iScript reverse transcriptase for one-step RT-PCR | 0.5 L
| Final reactions volume | 25 L |
| 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) |
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