Abstract
BackgroundHousekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development.Methodology/Principal FindingsWe applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed.ConclusionPutative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.
Highlights
Gene expression analyses are crucial for the discovery and characterization of the roles for known genes [1]
Our results showed that distinct combinations of internal controls fit for each experimental set in the case of quantitative RT-PCR (qRT-PCR) and that MAPK1 is a reliable loading control for Western blot
The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot
Summary
Gene expression analyses are crucial for the discovery and characterization of the roles for known genes [1]. Vertebrate retinas are composed of seven major cell types that are produced from multipotent progenitor cells [3,4]. During development, these progenitors expand through cell proliferation, commit to distinct cell types and exit the cell cycle to generate either retinal neurons or the Muller glia in an evolutionary conserved birth order [5]. We challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development
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