Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation.
Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation.
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DOI：
10.1186/1756-0500-4-443
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发表时间：
2011-10-25
影响因子：
1.8
通讯作者：
Pessler F
中科院分区：
文献类型：
--
作者：
Della Beffa C;Klawonn F;Menetski JP;Schumacher HR Jr;Pessler F
小鼠气囊膜代表了一种容易获得的组织，用于研究急性炎症中的基因调控。考虑到急性炎症可能影响分子参考基因的表达，我们评估了在尿酸盐结晶炎症的完整时间过程中气囊膜中甘油醛-3-磷酸脱氢酶（GAPDH）和脯氨酰肽基异构酶A（PPIA）的表达，并将结果与白细胞介素（IL）-1β和缺氧诱导因子（HIF）-1α的表达相关联。此外，我们的目的是确定替代的潜在参考基因。使用定制的微流控实时PCR阵列，在注射后1、4、9、18、27和50小时（h），在解剖的气袋膜中测定了包括GAPDH、PPIA、IL-1β和HIF-1α在内的96个基因的表达。尿酸单钠（MSU）晶体进入袋中。单因素方差分析用于检测整个时间过程中的差异基因表达。使用这些阵列上的基因作为方便的样品，（1）用生物统计方法和（2）使用geNorm软件工具寻找替代候选参考基因。囊袋白细胞在t = 9 h达到峰值，并在t = 50 h时下降。PPIA表达没有差异调节（p = 0.52，ANOVA）。相比之下，GAPDH mRNA在晶体注射后稳定地增加，在t = 18小时达到最大2.8倍增加（p = 0.0006，t检验），随后是IL-1β的显著诱导（max.，208-t = 4 h时的倍数，p = 8.4 × 10-5，t检验）和HIF-1α（最大值，6.6-t = 4 h时的倍数，p = 0.00025，t检验）。当Ct值针对GAPDH表达标准化时，15个基因被人为地鉴定为“显著调节”。生物统计方法和geNorm分析确定了候选参考基因的重叠集。两人都将PPIA列为最佳候选人，其次是细胞死亡防御者1（DAD 1）和高迁移率组B1（HMGB 1）。GAPDH mRNA表达在尿酸盐结晶炎症中上调，可能是由于炎症相关的缺氧。使用GAPDH mRNA进行分子标准化导致计算的靶mRNA表达中的显著伪影。PPIA和其他稳定表达的基因有望成为该模型中更合适的参考基因。
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.