UPA Perpustakaan Universitas Jember

Biological Procedures Online

Lei Liu - Personal Name
Frederik Mølgaard Nielsen - Personal Name
Simone Elkjær Riis - Personal Name
Jeppe Emmersen - Personal Name
Trine Fink - Personal Name
esper Østergaard Hjortdal - Personal Name
Chris Bath - Personal Name
Vladimir Zachar - Personal Name

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 procedure, while still preserving good discrimination between
the individual hLESC immunophenotypes.
Results: The 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.
Conclusion: In 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.

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Limbal stem cells
RNA integrity
Fluorescence-activated cell sorting
RNA sequencing
Intracellular staining
Ethanol

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Biological Procedures Online

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NPP. 3509212D1000001

Biological Procedures Online

Lei Liu - Personal NameFrederik Mølgaard Nielsen - Personal NameSimone Elkjær Riis - Personal NameJeppe Emmersen - Personal NameTrine Fink - Personal Nameesper Østergaard Hjortdal - Personal NameChris Bath - Personal NameVladimir Zachar - Personal Name

Series Title

Call Number

Publisher

Collation

Language

ISBN/ISSN

Classification

Content Type

Media Type

Carrier Type

Edition

Subject(s)

Specific Detail Info

Statement of Responsibility

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Lei Liu - Personal Name
Frederik Mølgaard Nielsen - Personal Name
Simone Elkjær Riis - Personal Name
Jeppe Emmersen - Personal Name
Trine Fink - Personal Name
esper Østergaard Hjortdal - Personal Name
Chris Bath - Personal Name
Vladimir Zachar - Personal Name