Crookenden M A, Walker C G, Kuhn-Sherlock B, Murray A, Dukkipati V S R, Heiser A, Roche J R
DairyNZ, c/o University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand; Institute of Vet, Animal and Biomedical Sciences, Massey University, Palmerston North 4442, New Zealand.
DairyNZ, c/o University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand.
J Dairy Sci. 2017 Aug;100(8):6763-6771. doi: 10.3168/jds.2016-12460. Epub 2017 Jun 16.
Reverse-transcription quantitative-PCR (RT-qPCR) is commonly used for assessing the cellular response to changes in physiologic and pathologic conditions. The selection of stable endogenous control genes is an important step of any RT-qPCR study, as expression can vary depending on the experimental environment. Our objective was to identify endogenous control genes in circulating neutrophils isolated from cows during the peripartum period. To do this, we used microfluidics gene expression arrays (Fluidigm, San Francisco, CA) for RT-qPCR analysis. Selection of the endogenous control genes was based on previous research investigating gene expression in neutrophils. The selected genes included ACTB, B2M, G6PD, GAPDH, GCH1, GOLGA5, OSBPL2, PGK1, RPL13A, RPL19, RPS9, SDHA, SMUG1, SNRPA, TBP, UXT, and YWHAZ. Four genes (GAPDH, GOLGA5, PGK1, and UXT) did not provide satisfactory quantification results using the selected method and were therefore excluded from the analyses. The suitability of the remaining 13 genes for use as endogenous control genes was assessed using geNorm and Normfinder. The gene pair with the greatest stability using geNorm was RPL13A and RPL19, whereas Normfinder ranked RPL19 and YWHAZ as the most stable pair. The 2 genes deemed most suitable for the experimental design were RPL19 and YWHAZ, which were selected for subsequent gene expression analysis. This study highlights that genes used as endogenous controls for relative quantification should be assessed on an experimental basis, even if the genes have been used in previous research.
逆转录定量聚合酶链反应（RT-qPCR）通常用于评估细胞对生理和病理条件变化的反应。选择稳定的内参基因是任何RT-qPCR研究的重要步骤，因为基因表达会因实验环境而异。我们的目标是鉴定围产期奶牛循环中性粒细胞中的内参基因。为此，我们使用微流控基因表达芯片（Fluidigm，加利福尼亚州旧金山）进行RT-qPCR分析。内参基因的选择基于先前对中性粒细胞基因表达的研究。所选基因包括ACTB、B2M、G6PD、GAPDH、GCH1、GOLGA5、OSBPL2、PGK1、RPL13A、RPL19、RPS9、SDHA、SMUG1、SNRPA、TBP、UXT和YWHAZ。使用所选方法时，四个基因（GAPDH、GOLGA5、PGK1和UXT）未提供满意的定量结果，因此被排除在分析之外。使用geNorm和Normfinder评估其余13个基因作为内参基因的适用性。使用geNorm时稳定性最高的基因对是RPL13A和RPL19，而Normfinder将RPL19和YWHAZ列为最稳定的基因对。被认为最适合实验设计的两个基因是RPL19和YWHAZ，它们被选用于后续的基因表达分析。这项研究强调，即使这些基因已在先前的研究中使用，用作相对定量内参的基因也应根据实验进行评估。
