Method for improved integrity of RNA isolated from Matrigel cultures
Method for improved integrity of RNA isolated from Matrigel cultures
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DOI：
10.1016/j.mex.2020.100966
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发表时间：
2020-01-01
期刊：
影响因子：
1.9
通讯作者：
Schmittgen, Thomas D.
中科院分区：
文献类型：
--
作者：
Da Silva, Lais;Bray, Julie K.;Schmittgen, Thomas D.
基质胶是一种市售的基质，来源于细胞外基质。基质胶广泛用于细胞培养实验，如原代胰腺腺泡转分化为导管上皮样细胞。在Matrigel上培养的细胞的基因表达分析期间出现困难，因为Matrigel中的残留RNA不仅会导致从Matrigel培养物分离的RNA的完整性差，而且会影响基因表达数据。我们在这里报告一个简单的方法，从人类或小鼠胰腺腺泡的原代培养物中去除基质胶。实验后，将培养物置于湿冰上以覆盖基质胶。然后将细胞和基质胶混合物低速离心以将胰腺细胞与存在于上清液中的基质胶溶液分离。从沉淀的细胞中分离的RNA具有高度的完整性，并且可以容易地用于基因表达分析，例如定量逆转录PCR。(C)2020年，任作家。由爱思唯尔公司出版
Matrigel is a commercially available substrate that is derived from the extracellular matrix. Matrigel is widely used in cell culture experiments such as the transdifferentiation of primary pancreatic acini to ductal epithelial-like cells. Difficulty arises during gene expression analysis for cells cultured on Matrigel because residual RNA in the Matrigel will not only contribute to the poor integrity of RNA isolated from Matrigel cultures, but also will impact the gene expression data. We report here a simple method of removing Matrigel from primary cultures of human or mouse pancreatic acini. Following the experiment, the cultures are placed on wet ice to liquefy the Matrigel. The cell and Matrigel mixture is then centrifuged at low speed to separate the pancreatic cells from the Matrigel solution that resides in the supernatant. RNA isolated from the pelleted cells has high integrity and may be readily used for gene expression analysis such as quantitative reverse transcription PCR. (C) 2020 The Authors. Published by Elsevier B.V.