Validation of reference genes in human testis and ejaculate
Validation of reference genes in human testis and ejaculate
复制标题
DOI：
10.1111/j.1439-0272.2010.01076.x
复制
发表时间：
2011-10-01
期刊：
影响因子：
2.4
通讯作者：
Steger, K.
中科院分区：
文献类型：
--
作者：
Cavalcanti, M. C. O.;Failling, K.;Steger, K.
研究人员对具有不同功能特征和表达模式的β-肌动蛋白（ACTB）、3-磷酸甘油醛脱氢酶（GAPD）、热休克蛋白1，β（HSPCB）和三磷酸腺苷5β亚基（ATP5B）进行了研究，作为基因表达研究的合适参考。我们应用实时 qRT-PCR 和统计分析确定了精液、冷冻保存以及固定和石蜡包埋的睾丸组织（来自生育力和生育力低下的男性）中四种参考基因的表达稳定性。通过使用 Wilcoxon-Mann-Whitney 检验，比较了可生育和低生育力供体之间每个基因的平均基因表达（平均 Ct 值）。我们没有观察到基因变异之间存在显着的统计差异。为了检测随机效应，我们使用分层模型的双向方差分析。结果显示先证者之间和先证者内重复之间没有显着的统计差异。综上所述，我们得出结论，ACTB、GAPD、HSPCB 和 ATP5B 在这些样本中表达存在差异，但这种差异在统计上并不显着。这一发现表明，所有这些基因都可以用于进一步研究射精和睾丸组织中的基因表达。因此，选择合适的参考基因对于特定的实验模型具有高度特异性，并且针对每种情况进行单独验证是至关重要的要求。
Beta-actin (ACTB), glyceraldehyde-3-phosphate-dehydrogenase (GAPD), Heat Shock Protein 1, beta (HSPCB) and Adenosine Triphosphate subunit 5 beta (ATP5B) with distinct functional characteristics and expression patterns were investigated as suitable references for gene expression studies. We determined the expression stability of the four reference genes in ejaculates, cryopreserved as well as fixed and paraffin-embedded testicular tissue (from fertile and subfertile men) applying real-time qRT-PCR and statistical analysis. The mean gene expressions (mean Ct value) were compared for each gene between the fertile and subfertile donors by using the Wilcoxon-Mann-Whitney test. We did not observe significant statistical differences between variability of genes. To detect random effects, we used the two-way analysis of variance with a hierarchical model. The results show no significant statistical differences between proband and repetition within the probands. Taken together, we concluded that ACTB, GAPD, HSPCB and ATP5B have a variable expression within these samples, but this variability is not statistically significant. This finding demonstrated that all these genes could be appropriated for further studies on gene expression in ejaculate and testis tissue. Therefore, the selection of the suitable reference genes is highly specific for a particular experimental model and validation for each situation, on an individual basis, is a crucial requirement.