Selective measurement of α smooth muscle actin: why β-actin can not be used as a housekeeping gene when tissue fibrosis occurs

IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology

BMC Molecular Biology Pub Date : 2017-04-27 DOI:10.1186/s12867-017-0089-9

Apor Veres-Székely, Domonkos Pap, Erna Sziksz, Eszter Jávorszky, Réka Rokonay, Rita Lippai, Kálmán Tory, Andrea Fekete, Tivadar Tulassay, Attila J. Szabó, Ádám Vannay

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引用次数: 31

Abstract

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 ?-actin from other actin isoforms.

Real-time PCRs using self-designed mouse, human and rat specific α-SMA or ?-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ?-actin, however ?-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 ?-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 ?-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ?-actin especially when fibrosis and thus increased expression of α-SMA is occur.

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α平滑肌肌动蛋白的选择性测量:为什么当组织纤维化发生时β肌动蛋白不能作为一个管家基因

包括慢性肾脏疾病在内的纤维增生性疾病的发病率正在迅速增加，并已成为世界范围内的一个主要公共卫生问题。纤维增生性疾病的特征是α平滑肌肌动蛋白(α- sma)的表达增加，该蛋白属于6种保守的肌动蛋白亚型家族，具有高度同源性。本研究的目的是开发实时pcr，明确区分α-SMA和?-肌动蛋白与其他肌动蛋白亚型。采用自行设计的小鼠、人、大鼠α-SMA或-肌动蛋白特异性引物对进行实时pcr，可扩增出小鼠、人、大鼠α-SMA或-肌动蛋白对应的人工DNA模板，但-肌动蛋白与内源性γ-细胞肌动蛋白发生交叉反应。我们已经证明，使用设计不当的文献引物对会显著影响pcr测量UUO小鼠肾脏中α-SMA或?-actin mRNA表达的结果。我们开发了一套精心设计的引物对和PCR条件，以选择性地测定小鼠、人或大鼠α-SMA和?-actin亚型的表达。我们在实验中证明了引物特异性的重要性，其中结果归一化为-肌动蛋白的表达，特别是当发生纤维化并因此增加α-SMA的表达时。

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来源期刊

BMC Molecular Biology 生物-生化与分子生物学

CiteScore

4.80

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： BMC Molecular Biology is an open access journal publishing original peer-reviewed research articles in all aspects of DNA and RNA in a cellular context, encompassing investigations of chromatin, replication, recombination, mutation, repair, transcription, translation and RNA processing and function.