Prevalence of fibroproliferative diseases, including chronic kidney disease is rapidly increasing and has become a major public health problem worldwide. Fibroproliferative diseases are characterized by increased expression of α smooth muscle actin (α-SMA) that belongs to the family of the six conserved actin isoforms showing high degree homology. The aim of the present study was to develop real-time PCRs that clearly discriminate α-SMA and ß-actin from other actin isoforms. Real-time PCRs using self-designed mouse, human and rat specific α-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping γ-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of α-SMA or ß-actin in the kidney of mice underwent UUO. We developed a set of carefully designed primer pairs and PCR conditions to selectively determine the expression of mouse, human or rat α-SMA and ß-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ß-actin especially when fibrosis and thus increased expression of α-SMA is occur.

中文翻译：

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α平滑肌肌动蛋白的选择性测量：为什么在组织纤维化发生时不能将β-肌动蛋白用作管家基因。

包括慢性肾脏疾病在内的纤维增生性疾病的流行正在迅速增加，已成为世界范围内的主要公共卫生问题。纤维增生性疾病的特征在于α平滑肌肌动蛋白（α-SMA）的表达增加，该α平滑肌肌动蛋白属于显示高度同源性的六个保守的肌动蛋白同工型家族。本研究的目的是开发可将α-SMA和ß-肌动蛋白与其他肌动蛋白同工型区分开的实时PCR。使用自行设计的小鼠，人类和大鼠特异性α-SMA或β-actin引物对进行实时PCR可以特异性扩增对应于小鼠，人类或大鼠α-SMA或β-actin的人工DNA模板，但是-肌动蛋白显示与管家的γ-细胞肌动蛋白发生交叉反应。我们已经表明，设计不当的文学引物对的使用会显着影响PCRs的测量结果，该PCRs测量接受UUO的小鼠肾脏中α-SMA或ß-actin的mRNA表达。我们开发了一组精心设计的引物对和PCR条件，以选择性确定小鼠，人类或大鼠α-SMA和ß-肌动蛋白同种型的表达。我们证明了引物特异性在实验中的重要性，在实验中将结果标准化为ß-肌动蛋白的表达，尤其是当发生纤维化并因此导致α-SMA表达增加时。