Pcr primers

Total RNA was extracted from renal tissues using Trizol according to the manufacturer's instructions (Takara, Japan). We obtained complementary DNA (cDNA) by reverse transcribing four micrograms of total RNA by using the PrimeScript RT Master Mix (Takara, Japan) as instructed. Real-time PCR amplifications were performed by using qPCR technique of SybrGreen assay on the ABI 7500 system (Applied Biosystems, USA). PCR primers (Takara, Japan) for all analyzed genes are as follows: tumor necrosis factor-α (TNF-α), amplicon size 122 bp, forward, 5′-GTG GAA CTG GCA GAA GAG GC-3′ and reverse, 5′-AGA CAG AAG AGC GTG GTG GC-3′; interleukin-1β (IL-1β), amplicon size 230 bp, forward, 5′-GCC CAT CCT CTG TGA CTC AT-3′ and reverse, 5′-AGG CCA CAG GTA TTT TGT CG-3′; COX-2, amplicon size 121 bp, sense: 5′-CCT GGT CTG ATG ATG TAT GC-3′; antisense: 5′-GTA TGA GTC TGC TGG TTT GG-3′; iNOS, amplicon size 108 bp, forward, 5′-TCC ATG ACT CCC AGC ACA-3′ and reverse, 5′-CCA TCT CCT GCA TTT CTT CC-3′; GAPDH, amplicon size 211 bp, forward, 5′-CAT CAA CGG GAA GCC CAT C-3′ and reverse, 5′-CTC GTG GTT CAC ACC CAT C-3′. PCR conditions were as follows: 94°C for 5 min; 35 cycles at 94°C for 40 s, 58°C for 40 s, and 72°C for 60 s; final elongation at 72°C for 10 min. The relative expression levels were calculated using the 2^−ΔΔCt method as reported.

Treatment with various drugs was added to logarithmic growth phase cells. The RNA from cells was extracted using the total RNA extraction kit [centrifugal columnar type; Tiangen Biotech (Beijing) Co., Ltd., Beijing, China]. The RNA was reverse transcribed into complementary DNA (cDNA) according to the manufacturer’s instructions. PCR primers were purchased from Takara Bio, Inc. (Shiga, Japan). The primers used for PCR amplification were: sense, 5′-AGACAGAAATGGCACCAC-3′ and antisense, 5′-AATGGGAGTCTTCTTTGG-3′ for CD44v6 (224-bp product); and sense, 5′-AATCCCATCACCATCTTCC-3′ and antisense, 5′-CATCACGCCACAGTTTCC-3′ for GAPDH (382-bp product). Reaction system of PCR (20 μl altogether) included 2 μl cDNA, 1 μl forward primer (10 μmol/l), 1 μl reverse primer (10 μmol/l), 25μl 2X TransTaq™ HiFi PCR SuperMix II and 21 μl ddH₂O. The conditions for PCR were as follows: predenaturation at 94°C for 5 min, 94°C for 30 sec, 50°C for 30 sec and 72°C for 30 sec, for 36 cycles; and elongation at 72°C for 5 min. PCR products were detected by 2% agarose gel. The band density was detected using the Bio-Rad Quantity One software (Bio-Rad, Hercules, CA, USA). The relative band density represented the relative expression levels of CD44v6 and was calculated using the formula:
$\text{Relative\hspace{0.17em}density\hspace{0.17em}of\hspace{0.17em}bands}=\text{CD}44\text{v}6\hspace{0.17em}\text{band\hspace{0.17em}density}/\text{GAPDH\hspace{0.17em}band\hspace{0.17em}density}$ .

Total RNAs were extracted from frozen tissue samples of patients with cervical cancer using Trizol reagent (Invitrogen, US) and treated by DNase / RNase-free Deionized water (Tiangen, Beijing, China). cycA was used as internal controls in SYBR^® Green Realtime PCR Master Mix-Plus kits (Toyobo, Osaka, Japan). The expression level of XRCC1 were amplified with PCR primers (Takara, Shanghai, China) and quantitative using the Quant Studio 6 Flex system (Applied Biosystems, Life Technologies, USA).