Stratagene mx3005p detector

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used for the extraction of total RNA from the tumor tissues according to the manufacturer’s protocol. Next, cDNA was synthesized from 2 μg total RNA using the PrimeScript^® first strand cDNA synthesis kit (Takara, Kusatsu, Japan). Actin was used as a constitutive control for normalization. cDNA was amplified using specific primers. qRT-PCR was performed using the SYBR premix ExTaq system (Takara, Kusatsu, Japan) and Stratagene Mx3005P detector (Agilent Technologies, Santa Clara, CA, USA). PCR conditions included one cycle of 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C, 45 s at 58 °C and 45 s at 72 °C. The fluorescent product was detected at the end of the 72 °C extension period. All samples were analyzed by three independent measurements. Primer pairs are listed in Table 1.

Total RNA was extracted from whole cells using TRIzol (Invitrogen, Carlsbad, CA, USA) and subsequently used to synthesize cDNA. Quantitative real-time polymerase chain reaction (PCR) was performed using the SYBR Premix ExTaq system (Takara, Shiga, Japan) and Stratagene Mx3005P detector (Agilent Technologies, Santa Clara, CA, USA). Table 1 shows the sequences of the primer pairs used.