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.

Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures

“…The Calibrated Normalized Relative Quantity (CNRQ) values depicted in the graph represent the normalized, relative expression levels for each mucin gene separately. These values are acquired through the normalization with four reference genes and their relation with the lowest expression level of a specific gel-forming mucin gene in all the samples [ 26 ]. As shown in Figure 2 , the relative mRNA expression of the four mucins drops significantly with time in culture (Fixed effect test of mixed effect model, p -value < 0.0001).…”

“…To obtain mRNA and intracellular proteins representing the profile of in vivo conjunctival cells, single cells were detached from their underlying connective tissue through an enzymatic dispase digestion protocol, as previously described [ 26 ]. The cellular pellet obtained was lysed using the RNeasy microkit (Qiagen), according to the manufacturer’s instructions, or using a protein lysis buffer, as specified in Section 4.6 .…”

“…A step-by-step protocol on the plating of bulbar conjunctiva was performed as previously described [ 26 ]. Briefly, 2 mm × 2 mm explants were cultured at the liquid air surface at 37 °C and 5% CO 2 to initiate outgrowth.…”

“…Assessment of gel-forming mucin expression (MUC2, MUC5AC, MUC5B, and MUC6) was performed as previously described in [ 26 ]. The mucin primers and corresponding template controls were purchased from Bio-Rad; qHsaCID0011696 (MUC2), qHsaCID0017663 (MUC5AC), qHsaCID0011690 (MUC5B), and qHsaCID0020103 (MUC6).…”

Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

“…Measuring gene expression is increasingly important for a diverse range of clinical applications [23][24][25][26] . Purification of RNA, an essential prerequisite for qPCR, removes other cellular components and data must be normalised based on the stable expression of endogenous control genes.…”