Optimization of Housekeeping Gene for Normalization of Quantitative Polymerase Chain Reaction Assay in Gastric Cancer Research
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摘要: 目的 寻找胃癌研究中较为恒定和适宜作为内参基因。方法 选取50例术后胃癌组织和癌旁组织，1株胃正常上皮细胞株和6株胃癌细胞株，分别提取组织和细胞总RNA。qRT-PCR（realtime quantitative reverse transcription-polymerase chain reaction）实时定量方法检测内参基因GAPDH（Glyceraldehyde 3-phosphate dehydrogenase）、ACTB（β-actin）、RPⅡ（RNA polymerase subunitⅡ）和18sRNA在上述两组样本中的表达量，对比四种基因表达的变化。选择肿瘤标志物癌胚抗原CEA（carcino-embryonic antigen）作为待测目的基因，分别用上述4种基因作为内参，用相对定量qRT-PCR的方法在上述组织和细胞中检测CEA的表达。使用geNorm软件分析最优化的内参基因。结果 与癌旁组织相比，对应癌组织中GAPDH、ACTB、RPⅡ的表达均发生明显表达上调（P<0.05），上调倍数分别约为（6.49±1.12）、（5.02±0.87）和（3.68±0.78）倍。而18sRNA在两种组织中未发生明显表达变化（P>0.05）。GAPDH、ACTB、RPⅡ和18sRNA在胃正常上皮细胞和胃癌细胞中表达水平未发生明显变化。用GAPDH、ACTB、RPⅡ作为内参基因时，CEAmRNA上升的倍数和比率均明显减小（P<0.05）。用18sRNA作为内参时，CEA在癌组织中表达明显上升，上升倍数为（2.87±0.56）倍，与癌旁组织相比差异有统计学意义（P<0.01）。结论 在各种组织和细胞中没有完全恒定的内参基因。18sRNA可作为 qRT-PCR实验检测胃组织基因表达最适宜的内参基因。Abstract: Objective To select a suitable housekeeping gene to be the internal reference gene for the investigation of gastric cancer tissues. Methods Fifty pairs of gastric cancer tissues and corresponding adjacent tissues were obtained from the patients with gastric carcinoma undergoing tylectomy. Total RNA was extracted using Trizol from the above tissues, gastric cancer cell lines and normal gastric epithelial immortalized cells. Absolute qRT-PCR was employed to detect the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin(ACTB), RNA polymerase subunit Ⅱ (RPⅡ) and 18sRNA in the gastric cancer tissues and cells. Relative qRT-PCR was used to detect the expression level of carcinoembryonic antigen(CEA) in gastric cancer cells and tissues with the GAPDH, 18sRNA, ACTB and RPⅡ as the housekeeping gene respectively. geNorm software was used to screen the most suitable reference gene for qRT-PCR. Results Comparing with corresponding adjacent tissues, GAPDH, ACTB and RPⅡ expression were obviously up-regulated by (6.49±1.12), (5.02±0.87) and (3.68±0.78) fold in cancer tissues (P<0.05). Yet 18sRNA had no obvious expression change in gastric cancer and corresponding adjacent tissues (P>0.05). The expression of GAPDH, ACTB, RPII and 18sRNA all showed no obvious changes in gastric cancer cell lines compared with normal gastric epithelial cells. Only when 18sRNA was used as the normalizing gene was CEA obviously elevated by (2.87±0.56) fold in gastric cancer tissues. Conclusion There is no universal reference gene in various tissues and cells. 18sRNA is stably expressed in gastric samples and could be used as the most optimization of Housekeeping Gene.
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Key words:
- Gastric carcinoma tissues /
- Gastric carcinoma cells /
- qRT-PCR /
- Internal reference gene /
- 18sRNA
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