Reference gene validation for qPCR in rat carotid body during postnatal development

Abstract

BackgroundThe carotid bodies are the main arterial oxygen chemoreceptors in mammals. Afferent neural output from the carotid bodies to brainstem respiratory and cardiovascular nuclei provides tonic input and mediates important protective responses to acute and chronic hypoxia. It is widely accepted that the selection of reference genes for mRNA normalization in quantitative real-time PCR must be validated for a given tissue and set of conditions. This is particularly important for studies in carotid body during early postnatal maturation as the arterial oxygen tension undergoes major changes from fetal to postnatal life, which may affect reference gene expression. In order to determine the most stable and suitable reference genes for the study of rat carotid body during development, six commonly used reference genes, β-actin, RPII (RNA polymerase II), PPIA (peptidyl-proyl-isomerase A), TBP (TATA-box binding protein), GAPDH, and 18s rRNA, were evaluated in two age groups (P0-1 and P14-16) under three environmental oxygen conditions (normoxia, chronic hypoxia and chronic hyperoxia) using the three most commonly used software programs, geNorm, NormFinder and BestKeeper.FindingsThe three programs produced similar results but the reference gene rankings were not identical between programs or experimental conditions. Overall, 18s rRNA was the least stable reference gene for carotid body and, when hyperoxia and/or hypoxia conditions were included, actin was similarly unstable.ConclusionsReference or housekeeping gene expression for qPCR studies of carotid body during postnatal development may vary with developmental stage and environmental conditions. Selection of the best reference gene or combination of reference genes for carotid body development studies should take environmental conditions into account. Two commonly used reference genes, 18s rRNA and actin, may be unsuitable for studies of carotid body maturation, especially if the study design includes altered oxygen conditions.

A Comprehensive Approach to Identify Reliable Reference Gene Candidates to Investigate the Link between Alcoholism and Endocrinology in Sprague-Dawley Rats

“…This is in agreement with other studies using carotid body from different SD strains under different oxygenation levels [39], oligodendrocyte cells from age-asynchronized Winstar rats treated with LXR agonist [40], male flinder rat hippocampus treated with MB [35], and liver of hypophysectomized male and female SD rats [41]. However, 18S rRNA was considered a good reference gene in Winstar rat livers treated with hepatotoxins [30], human livers with alcoholic liver disease [42], the uterus of ovarietomized C57BL6 mice treated with estradiol [36], and in liver and gonads of male and female fathead minnow fish [37].…”

Validation of Reference Genes for qPCR Analysis of Resistance Training and Androgenic Anabolic Steroids on Hypothalamus, Adrenal Gland and Fat Tissue

“…In our study the three software utilized in these analyses produced similar results but the order of the results was not identical and in some cases was considerably different, which corroborate the findings reported by other study [46]. This difference may be attributed to different mathematical models used in each program [24].…”

Identification of suitable reference genes for BDV-infected primary rat hippocampal neurons

“…The lowest M-value corresponds to the most stable reference gene, while the highest corresponds to the least stable one. In previous studies, an M-value <0.5 was set as a cut-off to assess gene stability in homogenous samples (23). GeNorm identified miR-101a and U87 (M-value, 0.3160; Table II) as the most stable pair-wise combination of reference genes for the experimental groups on day 12.…”