University of Zurich, Switzerland.
BMC Mol Biol. 2009 Dec 11;10:106. doi: 10.1186/1471-2199-10-106.
Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats.
RNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), beta-actin (ACTB), beta-2-microglobulin (B2M), beta-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones.
The reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed.
基因表达分析是当代研究的重要工具，实时 PCR 是定量转录水平的首选方法。对合适的参考基因进行共分析对于准确的表达归一化至关重要。参考基因的表达可能会有所不同，例如在物种或组织之间；因此，在进行表达研究之前，必须测试候选基因。家猫是医学研究和兽医领域的重要研究对象。本研究的目的是开发用于八种潜在参考基因的 TaqMan 实时 PCR 检测，并测试它们在包括来自健康猫的血液、淋巴、内分泌和胃肠道组织以及 FeLV 感染猫的肿瘤组织在内的猫样本中的适用性。
对组织中的 RNA 进行了优化提取，以在不使用 DNA 酶处理的情况下最大限度地减少基因组 DNA (gDNA) 污染。建立并优化了 v-abl Abelson 鼠白血病病毒致癌基因同源物 (ABL)、β-肌动蛋白 (ACTB)、β-2-微球蛋白 (B2M)、β-葡萄糖醛酸酶 (GUSB)、羟甲基胆烷合成酶 (HMBS)、次黄嘌呤磷酸核糖基转移酶 (HPRT)、核糖体蛋白 S7 (RPS7) 和色氨酸 5-单加氧酶激活蛋白 ζ 多肽 (YWHAZ) 的实时 PCR 检测。在所研究的八个基因中，有四个基因（ACTB、HPRT、RPS7 和 YWHAZ）存在假基因。这些检测与之前开发的用于猫甘油醛-3-磷酸脱氢酶 (GAPDH) 和通用 18S rRNA 基因的 TaqMan 检测一起进行了测试。十个候选参考基因的表达水平存在显著差异，最丰富的基因（18S rRNA）和最不丰富的基因（ABL、GUSB 和 HMBS）之间的表达差异约为 106 倍。geNorm 和 NormFinder 程序确定的稳定性存在显著差异。使用基于 ANOVA 的 NormFinder 程序，在所研究的组织中，RPS7 是最稳定的基因，其次是 ACTB 和 ABL；B2M、HPRT 和 18S rRNA 基因是最不稳定的基因。
在所研究的猫组织中，参考基因的表达稳定性差异很大。没有一个测试基因在所有组织中都是归一化的最佳选择。对于大多数组织，需要两到三个参考基因才能进行准确的归一化。本研究为根据分析的组织正确选择猫参考基因提供了必要的信息。
