Maintaining RNA Integrity for Transcriptomic Profiling of Ex Vivo Cultured Limbal Epithelial Stem Cells after Fluorescence-Activated Cell Sorting (FACS).

ABSTRACT:

Background

Transcriptomic profiling of ex vivo cultured human limbal epithelial stem cells (hLESCs) will foster better understanding of corneal physiology and novel treatment paradigms to limbal stem cell deficiency (LSCD). However, currently such profiling studies are hampered due to difficulties with producing sufficient amounts of intact mRNA for deep RNA sequencing (RNA-seq) from subpopulations sorted on the basis of co-expression of membrane and intracellular antigens by fluorescence-activated cell sorting (FACS).

Methods

To address this problem, we systematically analyzed the critical steps, and found that ethanol fixation together with optimized downstream procedures provided a pipeline that yielded high quality total RNA in amounts to readily support the RNA-seq proc

SUBMITTER: Liu L 

PROVIDER: S-EPMC5727887 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature