Selecting Appropriate Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Studies in Isolated and Cultured Ocular Surface Epithelia

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.

Figure 1

Schematic representation of the experimental culture set-up to obtain mRNA from isolated (day 0) and cultured (day 14) ocular surface epithelia. The steps to isolate human cadaveric donor tissue are in chronological order: the dissection of the inferior and superior bulbar conjunctival region (pink form, inferior region) and the removal of the ocular globe. After the isolation of inferior and superior keratolimbal biopsies from the ocular globe (green framework, inferior region), the corneolimbal epithelium can be trephined (green line = limbus, grey transparent surface = cornea). To determine the mRNA profile of in vivo limbal- (green), corneal- (grey), and conjunctival (pink) cells, the extracellular content of corneolimbal epithelium and conjunctival biopsies is enzymatically digested. The resulting single cell suspension is then lysed to allow mRNA collection. In parallel, limbal- and conjunctival explant cultures are initiated from keratolimbal and conjunctival biopsies. After a culture period of 14 days, confluent cultures undergo cell lysis to release their mRNA content. Ocular surface photograph © 2019 Zoë Dupon.

Figure 2

M-value of reference genes, determined by the geNorm software, in isolated (day 0) and/or cultured (day 14) limbal or conjunctival cells. The average expression stability is visualized during stepwise exclusion of the most unstable reference gene, leaving the most stably expressed genes on the right. The specific set of reference genes required to obtain an accurate normalization in each condition is underlined. The green lines represent the threshold of M-values that correspond to suitable reference genes in homogenous samples (M < 0.5) and heterogeneous samples (M < 1.0).