TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

The cultured A431 cells were treated with different concentrations of berberine (0, 25, 50, and 100 µg/mL), and incubated for an additional 4, 8, or 12 h. Total RNA was subsequently extracted from the cells using TRIzol reagent (Tiangen Biotech, Beijing, China). The RNA was reversetranscribed to cDNA. The cDNA samples were subjected to real-time PCR, using a Real Master Mix (SYBR Green) Kit (Tiangen Biotech). The following primers were used for PCR amplification: Bax, forward 5'-GGATGATTGCCGCCGT-3', reverse 5'-CCCAGTTGAAGTTGCCGT-3' (melting
temperature, Tm = 55°C); Bcl-2, forward 5'-TGCACCTGACGCCCTTCAC-3', reverse 5'-AGACA
GCCAGGAGAAATCAAACAG-3' (Tm = 55°C); Ezrin, forward 5'-ACGTCTGAGAATCAACAAGC-3',
reverse 5'-TTCTCCTCATAGTCCTGCAG-3' (Tm = 53°C); and GAPDH, forward 5'-GGTGAAGGTC
GGAGTCAACGGA-3', reverse 5'-GAGGGATCTCGCTCCTGGAAGA-3' (Tm = 61°C). The PCR conditions were set as follows: 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 s and Tm for
1 min, and 72°C for 7 min. The amplification reaction, PCR product detection, and data analyses were performed using the ABI Prism7900 Sequence Detection System (Applied Biosystems,
Waltham, MA, USA)

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Manufacturer protocol

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