Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation
1 Department of Cellular Proteomics, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany
2 Department of Infection Genetics, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany
3 Department of Epidemiology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany
4 Department of Computer Science, Ostfalia University of Applied Sciences, Salzdahlumer Str. 46/48, 38302 Wolfenbüttel, Germany
5 Department of Immunology, Merck Research Laboratories, 126 East Lincoln Avenue Rahway, NJ 07065-4646, USA
6 Division of Rheumatology, University of Pennsylvania, 3600 Spruce St, Philadelphia, PA 19104, USA
7 Division of Rheumatology, Philadelphia VA Medical Center, University and Woodland Avenues, Philadelphia, PA 19104, USA
8 University Children's Hospital, Faculty of Medicine "Carl-Gustav-Carus", Technical University Dresden, Fetscherstr. 74, 01307 Dresden, Germany
BMC Research Notes 2011, 4:443 doi:10.1186/1756-0500-4-443Published: 25 October 2011
The 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.
Using 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.
Pouch 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).
GAPDH 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.