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目的 建立科学的外周血有核细胞的冻存方法 ,为肿瘤的微转移逆转录PCR检测打好基础。方法 分离15例CEA分泌性肿瘤患者外周血有核细胞 ,分别用两种方法冻存 ,设为两组。对细胞进行总RNA的抽提 ,测定RNAOD值并行 1%琼脂糖凝胶电泳 ,进一步逆转录PCR反应、聚丙烯酰胺凝胶电泳分析最终产物。结果 经两种方法冻存的细胞 ,其总RNAOD值差异无显著性 ,电泳均见竹节样带。两组逆转录PCR结果一致 ,阳性率均为 66.7%。结论 在肿瘤的微转移逆转录PCR检测中 ,抽提组织或细胞总RNA是重要的环节 ,而对于后者来说 ,原始标本的冻存是很关键的。该实验中所采用的两种冻存方法都切实可行。
Objective To establish a scientific method of cryopreservation of peripheral blood nucleated cells and lay the foundation for the detection of tumor micrometastasis by reverse transcription PCR. Methods Peripheral blood nucleated cells from 15 patients with CEA secreting tumor were isolated and cryopreserved by two methods respectively, and were divided into two groups. Total RNA was extracted from the cells, RNAOD value was determined by 1% agarose gel electrophoresis, further reverse transcription reaction and polyacrylamide gel electrophoresis analysis of the final product. Results There was no significant difference in total RNA (OOD) between the two methods of cryopreservation of cells. The results of two sets of reverse transcription PCR were the same, the positive rates were 66.7%. CONCLUSIONS: Tissue or cell total RNA extraction is an important link in the detection of micrometastases by RT-PCR. For the latter, cryopreservation of the original specimen is crucial. The two freezing methods used in this experiment are feasible.