Superscript 3 platinum kit

Total RNA was extracted from cultured cells using Genelute Mammalian Total RNA kit (Sigma) according to the manufacturer’s protocol. RNA was quantified by spectrophotometric analysis and 1 μg of RNA was used to synthesize cDNA (SuperScript III Platinum Kit, Life Technologies). Real-time PCR analysis was performed using Stratagene MX3000P personal Q-PCR in the presence of SYBR Green. The PCR reagents were provided in SuperScript III Platinum Kit (Life Technologies), and the conditions were chosen according to manufacturer’s protocol. Primers were as follows: GAPDH forward primer: 5′-GGCCTCCAAGGAGTAAGACC-3′, reverse primer: 5′-AGGGGTCTACATGGCAACTG-3′; MCT1 forward primer: 5′-TTCGGGTGGCTCAGCTCCGT-3′, reverse primer: 5′-CCTCCTCCTTGGGCCCTCCA-3′; COX1 forward primer: 5′-TCCGCTACCATAATCATCGCT-3′, reverse primer 5′-CCGTGGAGTGTGGCGAGT-3′. Mean threshold cycle (Ct) values were determined by Stratagene software using three distinct amplification curves for each gene. Relative expression of the target gene was estimated using the formula: relative expression = 2×∆Ct, where ∆Ct = Ct (target gene) – Ct (GAPDH).

Total RNA was isolated from cervical cancer tissues and normal cervical tissues (n=18) using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The Superscript III Platinum Kit (Invitrogen) was used to reverse transcribe this total RNA into cDNA. Real-time PCR was performed with SYBR Green Master Mix (TaKaRa, Kyoto, Japan) using the following primers against HPIP: Forward, 5'-CCACCCACTTCTCTCAACTC-3'; Reverse, 5'-GATGAGGCTGCCAGGATA-3'. β-actin served as an internal reference; its expression was analyzed using the following primers: Forward, 5'-CTTAGTTGCGTTACACCCTTTCTTG-3'; Reverse, 5'-CTGTCACCTTCACCGTTCCAGTTT-3'. The experiments were performed in triplicate in the same reaction, and the results of the real-time quantitative RT-PCR experiments were analyzed using the 2^−ΔΔCt method.