Abstract
The introduction of tissue engineering has allowed scientists to push the boundaries and treat seriously damaged ocular surface epithelia. They have managed to do this through the development of biological substitutes that restore, maintain or improve tissue function. To ensure the generation of a therapeutically safe and effective graft, knowledge on the transcriptional profile of native and cultured ocular surface epithelia is of undeniable value. Gene expression studies are, however, only as reliable as their proper selection of internal reaction controls or reference genes. In this study, we determined the expression stability of a number of reference genes: 18s rRNA, ACTB, ATP5B, CyC1, EIF4A2, GAPDH, RPL13A, SDHA, TOP1, UBC, and YWHAZ in primary isolates as well as in ex vivo cultured ocular surface epithelia explants (day 0 and/or day 14). Expression stability of the reference genes was assessed with both the geNorm and NormFinder software that use a pairwise comparison and a model-based approach, respectively. Our results extend the general recommendation of using multiple reference genes for normalization purposes to our model systems and provide an overview of several references genes that are likely to be stable in similar culture protocols.
Highlights
Correlated with the presence of a healthy conjunctiva and tear film[10,11]
The following stable reference gene pairs have been determined to provide in a stable reference within the corresponding regions; hypoxanthine guanine phosphoribosyl transferase (HPRT1) - TATA-box binding protein (TBP) genes, β-glucuronidase (GUSB) - peptidylprolyl isomerase (PPIA), β2-microglobulin (B2M) - PPIA, and ribosomal protein large P0 (RPLP0) - phosphoglycerate kinase (PGK1)
Despite the majority of the M-values falling below the thresholds, we found that GAPDH and RPL13A lie above the 0.5 upper limit in cultured limbal stem cells
Summary
Correlated with the presence of a healthy conjunctiva and tear film[10,11]. it is essential to first normalize the tear film and rehabilitate the eyelid and fornix in patients with combined corneal and conjunctival pathology, using ex vivo cultured conjunctival tissue grafts, before proceeding with corneal reconstruction[10,11]. To assure regeneration and functional re-instatement of the cornea and conjunctiva, one aspect that needs to be controlled is the gene expression profile of the cultured limbal- and conjunctiva-derived cells. This profile should be identical to or resembling the profile of in vivo ocular surface epithelia to the utmost extent possible. Selecting a set of housekeeping genes that as a whole provides a stable reference, is of utmost importance to obtain representative results and to detect differences in expression profiles between conditions. No validation reports have yet been published on the accurate normalization potential of reference genes in cultured ocular surface epithelia
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