背景:从细胞中有效提取核酸和蛋白质(ENAP)是精确注释基因功能的先决条件,并已成为揭示生命奥秘的实验室常规。然而,细胞样本通常来自不同的培养皿,导致不可避免的实验误差,有时重复性差。
目的:探索一种提高ENAP工作效率的方法,最大限度地减少ENAP进程中的错误,提高后续实验结果的可靠性和可重复性。
方法:顺序分离RNA的方案,DNA,和使用RNAzol试剂来自相同培养的HepG2细胞的蛋白质在此呈现。第一步涉及培养HepG2细胞至指数期,然后依次分离RNA,DNA,和在第二步中来自相同培养细胞的蛋白质。在第三步中检测核酸和蛋白质的产量,它们的纯度和完整性在最后一步得到验证。
结果:该过程从开始到质量验证只需3-4d,效率很高。与现有的试剂盒和试剂相比,主要基于独立隔离,这种基于RNAzol试剂的方法的特点是连续分离RNA,DNA,和来自相同细胞的蛋白质,因此节省时间,成本低、效率高。
结论:RNA,DNA,用这种方法分离的蛋白质可用于逆转录聚合酶链反应,聚合酶链反应,和西方印迹,分别。
BACKGROUND: Efficient extraction of nucleic acids and proteins (ENAP) from cells is a prerequisite for precise annotation of gene function, and has become laboratory routine for revealing the mysteries of life. However, cell samples are often from different culture dishes, resulting in inevitable experimental errors and sometimes poor repeatability.
OBJECTIVE: To explore a method to improve the efficiency of ENAP, minimizing errors in ENAP processes, enhancing the reliability and repeatability of subsequent experimental results.
METHODS: A protocol for the sequential isolation of RNA, DNA, and proteins from the same cultured HepG2 cells using RNAzol reagent is presented here. The first step involves culturing HepG2 cells to the exponential phase, followed by the sequential isolation of RNA, DNA, and proteins from the same cultured cells in the second step. The yield of nucleic acids and proteins is detected in the third step, and their purity and integrity are verified in the last step.
RESULTS: The procedure takes as few as 3-4 d from the start to quality verification and is highly efficient. In contrast to the existing kits and reagents, which are primarily based on independent isolation, this RNAzol reagent-based method is characterized by the sequential isolation of RNA, DNA, and proteins from the same cells, and therefore saves time, and has low cost and high efficiency.
CONCLUSIONS: The RNA, DNA, and proteins isolated using this method can be used for reverse transcription-polymerase chain reaction, polymerase chain reaction, and western blotting, respectively.