Method for improved integrity of RNA isolated from Matrigel cultures

被引:12
作者
Da Silva, Lais [1 ]
Bray, Julie K. [2 ]
Bulut, Gamze [1 ]
Jiang, Jinmai [1 ]
Schmittgen, Thomas D. [1 ]
机构
[1] Univ Florida, Coll Pharm, Dept Pharmaceut, Gainesville, FL 32610 USA
[2] Univ Florida, Coll Med, Dept Pathol Immunol & Lab Med, Gainesville, FL USA
关键词
RNA integrity; Pancreas; Matrigel; Organoids; Extracellular matrix; Acinar ductal metaplasia;
D O I
10.1016/j.mex.2020.100966
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
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页数:5
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