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目的探讨单层培养细胞总RNA提取过程中先行细胞消化或离心富集对所提RNA质量是否产生影响。方法培养NGF诱导的PC12细胞,采用Trizol试剂提取RNA:离心组(A)先将贴壁生长细胞经胰酶消化、离心获得细胞沉淀,加入1 ml Trizol进行RNA提取;直接组(B)将培养液弃尽后直接加入Trizol试剂(1 ml/10 cm2瓶壁)进行RNA提取;均采用琼脂糖凝胶电泳分析RNA完整性,紫外分光光度仪测定RNA浓度和OD260/OD280比值。结果凝胶电泳结果显示,直接组出现3条清晰的条带(28S、18S和5S条带),离心组在电泳末端5S区出现粗大的条带。两组OD260/OD28比值相似,约为1.6。直接组组间样品间RNA浓度相差较大。结论采用离心法和直接法提取贴壁细胞总RNA,先行细胞离心富集增加了RNA降解机会,直接法提取的RNA质量较高,考虑对后续实验的影响,直接法更优于离心法。
Objective To investigate whether cell digestion or centrifugation enrichment during the extraction of total RNA from monolayer cultured cells affects the quality of RNA extracted. Methods Cultured PC12 cells induced by NGF and RNA extracted by Trizol reagent: Centrifugation group (A) The adherent cells were digested by trypsin and centrifuged to get the cell pellet. RNA was extracted by adding 1 ml Trizol. Direct group (B) Trizol reagent (1 ml / 10 cm2 bottle wall) was directly discarded after RNA was discarded. RNA integrity was analyzed by agarose gel electrophoresis. RNA concentration and OD260 / OD280 ratio were determined by ultraviolet spectrophotometer. Results The results of gel electrophoresis showed that there were three clear bands (28S, 18S and 5S bands) in the direct group and the thick bands in the 5S region of the electrophoresis group. OD260 / OD28 ratio of two groups is similar, about 1.6. There was a big difference in RNA concentration between the two groups. Conclusion Centrifugation and direct extraction of adherent cells total RNA, pre-enrichment of cells increased RNA degradation opportunities, RNA extracted by direct method of higher quality, consider the subsequent experiments, the direct method is better than the centrifugal method.