Abstract
Purpose :
The role of anemia on retinopathy of prematurity (ROP) is unclear; however, gene expression studies of phlebotomy induced anemia (PIA), its treatment with recombinant human erythropoietin (EPO) and oxygen induced retinopathy (OIR) may shed new light on the underlying molecular mechanism of ROP. Quantitative polymerase chain reaction (qPCR) is a sensitive technique for estimating the gene expression changes at the transcript level; however, there is no current consensus on reference genes for qPCR analysis of neonatal rat retinal tissue with PIA and OIR. We hypothesize that s18 is an unstable reference gene across these experimental conditions. In this interest, we carried out an evaluation of 8 commonly used reference genes, Ppia, Mapk1, Rplp0, S18, Hprt, Tbp, Rpp30 and Gapdh, and identified the most stable genes.
Methods :
4 groups of Sprague Dawley newborn rat pups with the following experimental conditions were generated: normoxia (control), normoxia with PIA, OIR, and OIR with PIA and EPO. The Penn et al. rat 50/10 model of OIR was used. PIA groups reached their hematocrit target of 15-20% (a 50% reduction from baseline) by P14-15. EPO groups received IP injections from P10-P20. At target timepoint, P14.5 or P20, pups were euthanized, followed by immediate whole retinal dissection. Retinas from 4-6 pups/group underwent RNA extraction, cDNA synthesis, and qPCR with 8 candidate reference genes selected from the literature. qPCR was run in duplicate. Cycles threshold (Ct) values were analyzed by RefFinder which combines ΔCt, geNorm, Normfinder, and BestKeeper algorithms to determine and rank stability of each reference gene.
Results :
The three most stable reference genes were Rpp30 (stabilization index (S.I.) 1.316), Tbp (S.I. 1.732) and Ppia (S.I. 2.828). S18 and Gapdh showed the lowest stability (Figure 1). Variation of reference gene Ct values is shown in Figure 2.
Conclusions :
Rpp30, Tbp and Ppia expression is least affected by experimental conditions of OIR, PIA and EPO administration, therefore suitable to use for qPCR data normalization across any of these experimental conditions.
This is a 2021 ARVO Annual Meeting abstract.