**2.7 The isolation of total RNA**

Total RNA was isolated from cells in the control and the extract treatment (0.1 mg/ml) group, for 48 h by using a total RNA isolation kit (Invitrogen PureLink Micro-to-Midi Total RNA Isolation Kit, Cat. No: 12183-180). For the determination of the amount of isolated total RNA (diluted 1: 200), spectrophotometer (Cintra 20, GBC) was used at 260 nm [36].

### **2.8 Reverse transcriptase (RT)-PCR**

A total of 21 μl RNase-free water was put in each PCR tube. In this mix, 25 μl of the 2x Reaction mix (0.4 mM MSO4 of each deoxynucleoside triphosphate) and 2 l of RT/Platinum ™ Taq Max were added. Then, 1l of *bcl-x, bik, mcl-1, bfl-1*, and β-*actin* (Takara BCL-2 familly, Cat no.6623) and 1l of total RNA of each experimental group was added to the tube for one-step RT-PCR (The Super Script™ One-Step RT-PCR Kit Invitrogen, Cat no. 10928-042).

Reverse transcription and amplification were performed in TC412 Techne. The RT-PCR profile used was as follows: a reverse transcription stage at 55°C for 30 min, followed by an initial denaturation stage at 94°C for 2 min. This was then followed by 40 amplification cycles at 94°C for 15 s, at 60°C for 30 s, and at 68°C for 1 min, and a final extension step at 68°C for 5 min. The resulting PCR products were visualized by electrophoresis through 1.8% agarose gel containing ethidium bromide and then visualized under UV light [37].

## **2.9 Statistical analysis**

Apoptotic index and cytotoxicity were analyzed by one-way ANOVA, followed by Dunnett's test for separate comparisons with the control group and the T-test for separate comparisons with each of the groups. Differences were considered significant at p < 0.05 (GraphPad Prism version 4.00, GraphPad Software, San Diego California USA).
