β-actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels
β-actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels
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DOI：
10.1136/thorax.57.9.765
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发表时间：
2002-09-01
期刊：
影响因子：
10
通讯作者：
Walters, EH
中科院分区：
文献类型：
--
作者：
Glare, EM;Divjak, M;Walters, EH
背景：利用逆转录-聚合酶链式反应(RT-PCR)来测量mRNA水平导致了β-肌动蛋白和GAPDH看家基因作为样本比较的分母的普遍使用。假设这些基因的表达保持不变，因此在处理和信号量化方面的变化是正常的。然而，已有文献表明，β-肌动蛋白和谷氨酸脱氢酶在S的表达随着细胞的增殖、活化和分化而上调。我们假设不同细胞型和不同激活状态的呼吸道样本有不同水平的GAPDH和β-肌动蛋白的表达。方法：采用竞争性RT-PCR方法检测26例正常对照和92例哮喘患者的BAL液细胞和支气管内膜活检组织中β-肌动蛋白、GAPDH和IL-2mRNA的表达水平。未使用吸入性皮质类固醇(ICS)的哮喘患者BAL液和活检组织中的GAPDHmRNA水平和BAL液细胞中的β-肌动蛋白mRNA水平均低于正常对照组和使用ICS的哮喘患者的10倍。活检标本中的β-肌动蛋白mRNA显示出相同的表达模式，但未使用ICS的哮喘患者与接受ICS治疗的哮喘患者没有显著差异。IL-2mRNA水平在受试者和治疗组之间没有差异，但当表达为与β-肌动蛋白的比率时，显示出显著差异。结论：在哮喘研究中，将β-肌动蛋白和GAPDH作为基因表达定量的分母可能会造成混淆。看家基因在用于这种定量的信使核糖核酸分析之前需要仔细的验证。
Background: The use of reverse transcription-polymerase chain reaction (RT-PCR) to measure mRNA levels has led to the common use of beta-actin and GAPDH housekeeping genes as denominators for comparison of samples. Expression of these genes is assumed to remain constant, so normalising for variations in processing and signal quantitation. However, it is well documented that beta-actin and GAPDH expression S upregulated with proliferation, activation, and differentiation. We hypothesised that airway samples which differ in their cellular profiles and activation status have different levels of expression of GAPDH and beta-actin,Methods: The mRNA for beta-actin, GAPDH, and interleukin (IL)-2 was measured in bronchoalveolar lavage (BAL) fluid cells and endobronchial biopsy tissue by competitive RT-PCR in a cross sectional study of 26 normal controls and 92 asthmatic subjects.Results: For both BAL fluid cells and biopsy tissue, asthmatics overall had reduced expression of GAPDH and beta-actin mRNA. In asthmatic subjects not using inhaled corticosteroids (ICS), GAPDH mRNA levels in both BAL fluid and biopsy tissue, and beta-actin mRNA in BAL fluid cells were 10 times lower than samples from both normal controls and from asthmatic subjects using ICS. beta-Actin mRNA in biopsy specimens showed the same pattern of expression, but asthmatic subjects not using ICS were not significantly different from those receiving ICS treatment. IL-2 mRNA levels did not differ between the subject or treatment groups but, when expressed as a ratio with beta-actin, significant differences were seen,Conclusions: beta-Actin and GAPDH used as denominators of gene expression quantitation in asthma research can cause confounding. Housekeeping genes need careful validation before their use in such quantitative mRNA assays.