survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma.
Bottom Line: Expression levels of survivin and glyceraldehyde-3-phosphate dehydrogenase mRNA were analysed quantitatively by real-time reverse transcriptase polymerase chain reaction (RT-PCR).Tumour-survivin/glyceraldehyde-3-phosphate dehydrogenase ratio did not correlate with histologic type, lymph node metastasis, and stage of tumours.In these patients, tumour-survivin mRNA expression was recognised as a good marker of cancer recurrence independently from tumour stage.
Affiliation: First Department of Surgery, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, Yonago 683-8504, Japan. firstname.lastname@example.orgShow MeSH
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Mentions: Quantification of gene expression was performed by real-time quantitative RT–PCR (Gene Amp® 5700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA, USA)), which uses the 5′ nuclease activity of Taq polymerase to detect PCR amplicons (Yajima et al, 1998; Mitas et al, 2001). The PCR solution (50 μl) was composed of 1 μl of cDNA solution, 5 pmol of the forward and reverse primers, 10 pmol of internal probe, and TaqMan Universal PCR Master Mix. PCR was carried out after incubation at 50°C for 2 min, denaturing at 95°C for 10 min, 45 cycles of 95°C for 15 s, and 61°C for 1 min. Experiments were performed in duplicate for each data point. For each reaction tube, the fluorescence signal of the reporter dye (FAM) was divided by the fluorescence signal of the passive reference dye (TAMRA) to obtain a ratio defined as the normalised reporter signal (Rn). The threshold line was set at an Rn of 0.05 (10 standard deviations above the mean of the baseline fluorescence emission calculated from cycles 3–20) (Yajima et al, 1998). The point at which the amplification plot crossed this threshold was defined as Ct, which represented the cycle number at this point. Standard curves for survivin, and GAPDH were generated using serial dilution (containing 160, 80, 40, 20 and 10 ng) of total RNA derived from the EC-GI-10 cell line. The plots represent the log of the input amount (log ng of total starting RNA) as the x-axis and Ct as the y-axis. Equations were derived from the lines of the calibration curves (Yajima et al, 1998). The formulas for survivin and GAPDH were as follows: survivin, y=28.6–3.6x (r2=0.998) and GAPDH, y=26.3–4.5x (r2=0.991) (Figure 1Figure 1
Affiliation: First Department of Surgery, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, Yonago 683-8504, Japan. email@example.com