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目的:比较改进SDS-Phenol法与常用的AGPC法提取细胞之总RNA优缺点。方法:同时采用两种方法对同批稳定培养的一株淋巴瘤细胞系进行总RNA提取,以收获RNA的产量和纯度为比较指标,并用甲醛变性电泳验证SDS-Phenol法提取RNA的完整性。结果:改进SDS-Phenol法可从1×104细胞中提取(1.93±0.20)μg的总RNA,是AGPC法的两倍以上。细胞数在1×105~100×105范围内时其RNA产量与细胞数量呈线性相关。5×105以上细胞所提取的RNA,A260/280在1.78~1.83之间,无蛋白质,纯度平均在90%以上,甲醛变性电泳示RNA完整无降解。5×106细胞可收获RNA(55.34±3.02)μg,变异系数5.45%。结论:改进SDS-Phenol法优于AGPC法,有快速、简便和高效的特点,值得推广应用。
OBJECTIVE: To compare the advantages and disadvantages of using modified SDS-Phenol and conventional AGPC methods to extract total RNA from cells. Methods: Total RNA was extracted from one lymphoma cell line stably cultured in the same batch by two methods. The yield and purity of the harvested RNA were compared to evaluate the integrity of RNA extracted by SDS-Phenol. Results: The modified SDS-Phenol method can extract 1.93 ± 0.20 μg total RNA from 1 × 104 cells, which is more than twice that of AGPC method. The number of cells in the range of 1 × 105 ~ 100 × 105 RNA yield and cell number was linearly correlated. The RNA extracted from cells with a size of more than 5 × 105 had an A260 / 280 of between 1.78 and 1.83, no protein, and an average purity of over 90%. Formaldehyde denaturing electrophoresis showed that RNA was intact without degradation. 5 × 106 cells harvested RNA (55.34 ± 3.02) μg, coefficient of variation of 5.45%. Conclusion: The improved SDS-Phenol method is superior to AGPC method, which is fast, simple and efficient, which is worth popularizing.