评估用于猪肺泡巨噬细胞响应脂多糖和脂磷壁酸的基因表达研究的合适内参基因。

Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA.

作者信息

Cinar Mehmet Ulas, Islam Mohammad Ariful, Uddin Muhammad Jasim, Tholen Ernst, Tesfaye Dawit, Looft Christian, Schellander Karl

机构信息

Institute of Animal Sciences, Unit of Animal Breeding and Husbandry, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany.

出版信息

BMC Res Notes. 2012 Feb 18;5:107. doi: 10.1186/1756-0500-5-107.

BACKGROUND

To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs) is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in pigs are relatively rare and have never been performed in porcine alveolar macrophages (AMs). In this study, expression stability of putative housekeeping genes were identified in the porcine AMs in response to the stimulation with two pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Three different algorithms (geNorm, Normfinder and BestKeeper) were applied to assess the stability of HKGs.

RESULTS

The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by qRT-PCR in AMs that were stimulated by LPS and LTA in vitro. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.0001). geNorm software revealed that SDHA, B2M and RPL4 showed a high expression stability in the irrespective to the stimulation group, while SDHA, YWHAZ and RPL4 showed high stability in non-stimulated control group. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that SDHA was the most stable gene in all the groups. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.

CONCLUSIONS

There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms. In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of the animals.

背景

为获得可靠的定量实时PCR数据，需要相对于稳定的管家基因（HKGs）进行标准化。然而，在实际中，已发现“典型”管家基因的表达水平在不同组织和不同实验条件下会有所变化。迄今为止，猪中参考基因的验证研究相对较少，且从未在猪肺泡巨噬细胞（AMs）中进行过。在本研究中，在猪AMs中鉴定了假定管家基因的表达稳定性，以响应两种病原体相关分子模式（PAMPs）脂多糖（LPS）和脂磷壁酸（LTA）的刺激。应用三种不同算法（geNorm、Normfinder和BestKeeper）评估HKGs的稳定性。

结果

通过qRT-PCR在体外受LPS和LTA刺激的AMs中测定了九个常用参考基因（B2M、BLM、GAPDH、HPRT1、PPIA、RPL4、SDHA、TBP和YWHAZ）的mRNA表达稳定性。发现所有基因的mRNA表达水平受刺激类型和刺激持续时间的影响（P < 0.0001）。geNorm软件显示，无论刺激组如何，SDHA、B2M和RPL4均表现出高表达稳定性，而在未刺激的对照组中，SDHA、YWHAZ和RPL4表现出高稳定性。在所有情况下，GAPDH在geNorm中表现出最低的稳定性。NormFinder显示SDHA是所有组中最稳定的基因。此外，geNorm软件表明，三个最稳定基因的几何平均值将是基因表达研究准确标准化的合适组合。