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

Abstract

BackgroundTranscriptomic 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).MethodsTo 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 procedure, while still preserving good discrimination between the individual hLESC immunophenotypes.ResultsThe average RNA integrity number (RIN) was 7.7 ± 0.4, and the average yield was 4.6 ± 1.7 pg of RNA per cell. The sequencing analysis of the isolated RNA produced high quality data with more than 70% of read pairs mapping uniformly to the reference genome and 80% of bases with a Phred score of at least 30.ConclusionIn this study, we developed a reliable FACS-based procedure using ethanol as a fixative that would support accurate isolation of limbal epithelial progenitor subpopulations along with RNA yield and quality sufficient to enable deep transcriptomic profiling.Electronic supplementary materialThe online version of this article (10.1186/s12575-017-0065-2) contains supplementary material, which is available to authorized users.

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells

“…Before sequencing, the RNA quality was analyzed using parameters described previously , and the values pertinent to individual populations are shown in Supporting Information Table S2. Although the RNA yield varied broadly, up to 2.8‐fold between the highest and lowest values, the RIN appeared consistent and sufficiently high (7.7 ± 0.4, n = 4) to meet the requirements of the protocol.…”

“…The experimental set up was previously optimized to reveal markers pertinent to this study using a set of directly labeled antibodies and to determine the sorting thresholds using matching isotype controls for p63 and CK3 and fluorescence minus one (FMO) for ABCB5 . All buffers used in staining and subsequent FACS sorting were sPBS based, supplied with 50% Accumax (Sigma‐Aldrich) and 25 mM HEPES (Life Technologies) to prevent cell clumping and to maintain a proper pH range.…”

“…To overcome these limitations, we have in this study combined our earlier optimized pipeline for high quality transcripts from human limbal epithelial cellular subpopulations sorted by fluorescence‐activated cell sorting (FACS) with discovery based next‐generation sequencing. By using this strategy, we have as the first conducted a biomarker‐oriented comparative transcriptome analysis of cultured and sorted hLESCs, and by this sought to refute or confirm the use of p63 and ABCB5 as surrogate markers for hLESCs.…”

Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials

“…For example, antibodies against intracellular antigens were not tested since this necessitates cell fixation and/or permeabilization for FACS. However, others have reported that efficient RNA recovery from fixed cells may be feasible [34–36]. Although most cells and biomaterials were fresh, our experiments did not control for age and variability in storage conditions, which may have affected our results.…”