基于核酸定量的核质分离质控方案

目的:拟建立一种方便快捷、经济有效的细胞核质分离鉴定方法。方法:本研究从DNA水平进行核质分离鉴定,选择GAPDH、ND1分别作为细胞核和细胞质标志基因,并根据GAPDH及ND1序列保守区设计引物,基于荧光定量PCR方法定性检测核质分离的效果。随后将本鉴定方法应用于其他种类细胞(BEAS-2B细胞、GT1-7细胞)及组织(小鼠心脏、肝脏、大脑)的核质分离鉴定。结果:在293T细胞应用该鉴定方法鉴定核质分离效果,结果显示:GAPDH、ND1在核组、质组中的含量存在明显差异,其中核标志基因GAPDH在细胞核中的比例达到了95%以上,质标志基因ND1在细胞质中的比例也达到了90%左右,这与从RNA水平及蛋白水平鉴定核质分离的结果一致。在其他种类的细胞(BEAS-2B细胞、GT1-7细胞)及组织(小鼠心脏、肝脏、大脑)应用该方法结果显示:细胞核组分中质标志基因ND1含量比293T细胞的有所增加,但仍可以实现核质分离鉴定。结论:本研究所建立的核质分离质控方法可以实现从DNA水平进行核质分离的鉴定,该方法更加经济、快捷。

Nucleocytoplasmic Separation Quality Control Scheme Based on Nucleic Acid Quantification

ABSTRACT Objective: It is proposed to establish a convenient, rapid and cost-effective method for identification of nucleocytoplasmic separation. Methods: In this study, identification of nucleocytoplasmic separation were performed at the DNA level. GAPDH and ND1 were selected as nuclear and cytoplasmic marker genes respectively, moreover primers were designed according to the conserved regions of GAPDH and ND1 sequences. The effect of nucleocytoplasmic separation was determined by Real-time quantitative PCR. The identification method was applied to other types of cells (BEAS-2B cells, GT1-7 cells) and tissues (mouse heart, liver, brain). Results: The method was used to identify the effect of nucleocytoplasmic separation in 293T cells. The results showed that there were significant differences in the content of GAPDH and ND1 in the nuclear and cytoplasm groups. And the ratio of nuclear marker gene GAPDH in the nucleus was over 95%, the proportion of cytoplasm marker gene ND1 in the cytoplasm also reached about 90%, which was consistent with the results of identification from RNA and protein levels. The results of this method in other kinds of cells (BEAS-2B, GT1-7) and tissues (mouse heart, liver, brain) showed that the content of ND1 in the nuclear component was increased compared with that of 293T cells. However, identification of nucleocytoplasmic separation can still be achieved in those kinds of cells or tissues. Conclusion: We create a more econom