Uv gel imaging system

Semi-quantitative determination of E2F1,CDKN1A,MKI67,CCND1 and Raf1 gene expression was assessed and compared with the results of the gene expression profile chip. Total RNA was extracted from the tumor tissues of 3 mg/kg FNC and negative control groups using Trizol (ComWin, Beijing, China). Next, 1 μg of RNA was used to synthesize cDNA using ReverTra Ace (Toyobo, Japan) according to the manufacturer’s protocol. The primers used for RT-PCR are presented in Table 1. Human GAPDH was used as an internal control to normalize the mRNA levels. PCR amplification was carried out in a 50-μl reaction mixture with 25 μl of 2×Taq Master Mix (ComWin, Beijing, China), 19 μl of ddH2O, 2 μl (10μM) each of forward and reverse primers and 2 μl of cDNA. The PCR conditions included an initial denaturation at 94°C for 2 min followed by 40 amplification cycles consisting of denaturation for 30 sec at 94°C, annealing for 30 sec at 60°C and extension for 30 sec at 72°C, followed by a final extension for 2 min at 72°C. The annealing temperature was 60°C for GAPDH. PCR products were fractionated on 2% agarose gels and were photographed with a UV gel imaging system (Kodak, USA) using ImageJ software to analyze the gray values. Each assay was repeated three times.

Total RNA was extracted from PTC tissues and cell lines using the Trizol reagent (Invitrogen, USA). Total RNA (1 μg) was reverse-transcribed to cDNA using PrimeScript™ II kit (Takara, Japan) according to the manufacturer' instructions. One μl of the resulting cDNA was used as the template for PCR in a 25-μl reaction volume containing 12.5 μl Premix Taq (Takara, Japan), 1 μl of each primer, and 5 μl ddH₂O. The β-actin mRNA level was used for normalization. PCR primer sequences are as follows—for β-actin: 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′(forward) and 5′-TGGCACCCAGCACAATGAA-3′(reverse); for CCDC67: 5′-GCAGCTCTGAAATTCCTCGT-3′ (forward) and 5′-TTGGTTGATCTTGCATCACTG-3′ (reverse). The PCR cycles were performed as follows: one cycle at 95°C for 3 min, 40 cycles at 95°C for 30s, 60°C for 30s and 72°C for 30s, and one final cycle at 72°C for 10 min, followed by cooling to 4°C. Amplification products were separated on 3% agarose gels and photographed with a UV gel imaging system (Kodak, USA). Imagemaster DVS system was used to determine the relative mean gray values (A) of the target product and β-actin. Expression index (I) of the target product was calculated using the formula of I = A product/A β-actin as previously described [34 (link)].