Abstract
BackgroundThe murine air pouch membrane represents an easily accessible tissue for studies on gene regulation in acute inflammation. Considering that acute inflammation may affect expression of molecular reference genes, we evaluated the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and prolylpeptidyl isomerase A (PPIA) in the air pouch membrane during a complete time course of urate crystal inflammation and correlated the results with expression of interleukin (IL)-1β and hypoxia inducible factor (HIF)-1α. In addition, we aimed to identify alternate potential reference genes.MethodsUsing custom microfluidic real-time PCR arrays, the expression of 96 genes including GAPDH, PPIA, IL-1β, and HIF-1α was determined in dissected air pouch membranes 1, 4, 9, 18, 27, and 50 hours (h) after injecting monosodium urate (MSU) crystals into the pouch. One-way ANOVA was used to detect differential gene expression throughout the time course. Using the genes on these arrays as a convenience sample, alternate candidate reference genes were sought (1) with a biostatistical approach and (2) using the geNorm software tool.ResultsPouch leukocytes peaked at t = 9h and declined toward t = 50h. PPIA expression was not differentially regulated (p = 0.52, ANOVA). In contrast, GAPDH mRNA increased steadily after crystal injection, reaching a maximal 2.8-fold increase at t = 18h (p = 0.0006, t test), which followed a marked induction of IL-1β (max., 208-fold at t = 4h, p = 8.4 × 10-5, t test) and HIF-1α (max., 6.6-fold at t = 4h, p = 0.00025, t test). Fifteen genes were artifactually identified as "significantly regulated" when Ct values were normalized against GAPDH expression. The biostatistical approach and the geNorm analysis identified overlapping sets of candidate reference genes. Both ranked PPIA as the best candidate, followed by defender against cell death 1 (DAD1) and high-mobility group B1 (HMGB1).ConclusionsGAPDH mRNA expression is up-regulated in urate crystal inflammation, possibly due to inflammation-associated hypoxia. Using GAPDH mRNA for molecular normalization resulted in significant artifacts in the calculated expression of the target mRNAs. PPIA and other stably expressed genes promise to be more appropriate reference genes in this model.
Highlights
The murine air pouch membrane represents an accessible tissue for studies on gene regulation in acute inflammation
Hypoxia can induce glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, likely because binding of a complex of the inducible a subunit and the constitutively expressed b subunit of hypoxia inducible factor (HIF)-1 to a hypoxia response element (HRE) in the GAPDH promoter region can increase transcription of this gene [4,5,6]
Up-regulation of GAPDH, but not prolylpeptidyl isomerase A (PPIA), mRNA in urate crystal inflammation Quantification of pouch exudate leukocyte counts, the gold standard for measuring inflammation in this model, revealed a rapid evolution of inflammation that peaked at t = 9h and returned to near normal by t = 50h (Figure 2A)
Summary
The murine air pouch membrane represents an accessible tissue for studies on gene regulation in acute inflammation. Prolylpeptidyl isomerase A (PPIA) as potential reference genes GAPDH is often used for molecular normalization of gene expression data from microarrays or real-time reverse transcriptase polymerase chain reactions (qPCR). This is based on the assumption that expression of this “housekeeping gene” does not change much during the life cycle of most cells and can be used as a relatively constant reference signal. There is reason to suspect that it, too, may be a suboptimal reference gene in studies on inflammation
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