小鼠哮喘模型颈静脉-结状神经节内参基因的筛选
Selection of reference genes for expression studies in the jugular-nodose ganglion of a mouse model of asthma
目的 观察12个常用看家基因在正常小鼠和哮喘小鼠颈静脉-结状神经节的表达变化，选择合适的内参基因为进一步基因表达分析研究得到可信数据提供依据。 方法 雌性BALB/c小鼠随机分为哮喘模型组和对照组，制备哮喘模型，用实时荧光定量PCR技术，经geNorm及Normfinder程序分析，评价了12个常用看家基因CYC1、UBC、RPL13、YWHAZ、18srRNA、B2M、SDHA、ATP5B、CANX、 ACTB、EIF4A2和GAPDH在小鼠颈-结状神经节的表达稳定性。 结果 12个候选看家基因在小鼠神经节的表达差异较大，其中GAPDH、18srRNA、CANX、ACTB和EIF4A2表达相对稳定，而CYC1、UBC和RPL13的表达在两组小鼠神经节变化幅度较大，结合两种分析证实GAPDH是其中最稳定的内参基因。 结论 本研究结果提示GAPDH是研究哮喘小鼠和正常小鼠颈静脉-结状神经节基因表达的最佳内参基因，同时也为进一步认识在不同实验模型确定适宜内参基因的重要性提供依据。
Objective To investigate the expression stability of 12 common housekeeping genes (HKGs) in jugular-nodose ganglion complex (JNC) from control and asthmatic mice. Methods Female BALB/c mice were randomly divided into control group and asthma group. Expression levels of 12 candidate HKGs, CYC1, UBC, RPL13, YWHAZ, 18srRNA, B2M, SDHA, ATP5B, CANX, ACTB, EIF4A2 and GAPDH, were measured by quantitative real-time PCR in JNCs from 5 normal mice and 5 asthmatic mice. Gene expression stability was subsequently determined using geNorm and Normfinder applets. Results GAPDH, 18srRNA, CANX, ACTB and EIF4A2 were relative stably expressed. However, the expression levels of CYC1, UBC and RPL13 were significantly different between the two groups. GAPDH was ranked the most stable by geNorm and Normfinder. Conclusions The results suggest that GAPDH is the most suitable housekeeping gene analyzed for expression studies in JNCs from normal and asthmatic mice, and provide the evidence of the importance of validating housekeeping genes for different experimental model.
看家基因 / 哮喘模型 / 颈静脉-结状神经节 / 实时荧光定量PCR / geNorm / Normfinder
Housekeeping genes / Asthma model / Jugular-nodose ganglion complex / Quantitative real-time PCR / GeNorm / Normfinder
[1] Dinh QT, Klapp BF, Fischer A. Airway sensory nerve and tachykinins in asthma and COPD
[J]. Pneumologie, 2006,60(2):80-85.
[2] Thellin O, Zorzi W, Lakaye B, et al. Housekeeping genes as internal standards: use and limits
[J]. J Biotechnol, 1999,75(2-3):291-295.
[3] Selvey S, Thompson EW, Matthaei K, et al. β-Actin an unsuitable internal control for RT-PCR
[J]. Mol Cell Probes, 2001,15(5):307-311.
[4] Glare EM, Divjak M, Bailey MJ, et al. β-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalizing mRNA levels
[J]. Thorax, 2002,57(9):765-770.
[5] Herz U, Braun A, Ruckert R, et al. Various immunological phenotypes are associated with increased airway responsiveness
[J]. Clin Exp Allergy, 1998,28(5):625-34.
[6] Hahn C, Islamian AP, Renz H, et al. Airway epithelial cells produce neurotrophins and promote the survival of eosinophils during allergic airway in?ammation
[J]. J Allergy Clin Immunol, 2006(4):787-794.
[7] Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes
[J]. Genome Biol, 2002,3(7):research0034.
[8] Andersen CL, Jensen JL, Orntoft TF. Normalization of real-time quantitative reverse transcription-PCR data：a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets
[J]. Cancer research, 2004, 64(15):5245-5250.
[9] 陈凤花, 王琳. 实时荧光定量 RT-PCR内参基因的选择
[J]. 临床检验杂志, 2005,23(5):393-395.
[10]He JQ, Sandford AJ, Wang IM, et al. Selection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells
[J]. Eur Respir J, 2008,32(3):755-762.
[11]吕艺, 刘淑红, 葛学铭, 等. 中枢和外周神经损伤后胞体区看家基因表达的变化
[J]. 感染、炎症、修复, 2002,3(4):212-214.
[12]Rhinn H, Marchand-Leroux C, Croci N, et al. Housekeeping while brain's storming Validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury
[J]. BMC Molecular Biology, 2008,9:62-73.
广东省自然科学基金( 5006052)
京ICP备08002354号-3/
| 〈 |
|
〉 |
