不同饲料硒水平饲养大鼠的多组织内参基因筛选
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摘要
目的筛选不同饲料硒(selenium,Se)水平饲喂大鼠时,不同组织中稳定表达的内参基因(reference genes,RGs)。方法 24只断乳雄性SD大鼠在缺Se饲养5周后,随机均分为4组,分别以Se含量<0.01、0.25、3、5 mg/kg饲料饲喂4周后处死,取肝、睾丸、骨骼肌、脂肪组织等样品待检。以荧光定量PCR检测Actb、Atp5f1、B2m、Gapdh、Gusb、Hprt、Pgk1、Ppia、Rplp2、Rps18、Tbp、Ywhaz等12个候选内参基因的mRNA水平,以geNorm、NormFinder、BestKeeper、Delta CT和RefFinder等方法对其表达稳定性进行评价。结果各组织中稳定性排名前4的内参基因是:肝中Ppia>Atp5f1>Rplp2>Hprt;睾丸中Ywhaz>Atp5f1>Rplp2>Ppia;骨骼肌中Tbp>Ppia>B2m>Rps18;脂肪组织中Hprt>Tbp>Atp5f1>Pgk1;综合4种组织,则Rps18>Hprt>Rplp2>Atp5f1。结论分析不同饲料Se水平饲养大鼠的目标基因表达水平时,应根据组织类型选择适宜的内参基因。
OBJECTIVE To screen for the most stable reference genes(RGs) in various tissues of rats fed at different dietary concentrations of selenium(Se). METHODS Twenty-four weaning male SD rats were fed Se deficient diet for 5 weeks, and then randomly divided into 4 groups for<0.01, 0.25, 3 and 5 mg Se/kg diet feeding, respectively. After 4 weeks, animals were sacrificed for sample collection of liver, testis, muscle and fat tissue. Twelve candidate RGs of Actb, Atp5f1, B2m, Gapdh, Gusb, Hprt, Pgk1, Ppia, Rplp2, Rps18, Tbp and Ywhaz were tested for their quantitative cycle numbers of mRNA abundances with the quantitative PCR method. The stabilities of the candidate RGs were evaluated by the arithmetic packages of geNorm, NormFinder, BestKeeper, Delta CT and RefFinder. RESULTS The top 4 most stable RGs were Ppia >Atp5f1 > Rplp2 >Hprt in liver; Ywhaz > Atp5f1 >Rplp2> Ppia in testis; Tbp > Ppia > B2m > Rps18 in muscle; Hprt>Tbp >Atp5f1>Pgk1 in fat tissue; and Rps18>Hprt> Rplp2>Atp5f1 when all the 4 tissues combined for analysis. CONCLUSION To analyze the expressions of the target genes in rats fed different concentrations of dietary Se, the best RGs should be selected depending on the tissue types.
OBJECTIVE To screen for the most stable reference genes(RGs) in various tissues of rats fed at different dietary concentrations of selenium(Se). METHODS Twenty-four weaning male SD rats were fed Se deficient diet for 5 weeks, and then randomly divided into 4 groups for<0.01, 0.25, 3 and 5 mg Se/kg diet feeding, respectively. After 4 weeks, animals were sacrificed for sample collection of liver, testis, muscle and fat tissue. Twelve candidate RGs of Actb, Atp5f1, B2m, Gapdh, Gusb, Hprt, Pgk1, Ppia, Rplp2, Rps18, Tbp and Ywhaz were tested for their quantitative cycle numbers of mRNA abundances with the quantitative PCR method. The stabilities of the candidate RGs were evaluated by the arithmetic packages of geNorm, NormFinder, BestKeeper, Delta CT and RefFinder. RESULTS The top 4 most stable RGs were Ppia >Atp5f1 > Rplp2 >Hprt in liver; Ywhaz > Atp5f1 >Rplp2> Ppia in testis; Tbp > Ppia > B2m > Rps18 in muscle; Hprt>Tbp >Atp5f1>Pgk1 in fat tissue; and Rps18>Hprt> Rplp2>Atp5f1 when all the 4 tissues combined for analysis. CONCLUSION To analyze the expressions of the target genes in rats fed different concentrations of dietary Se, the best RGs should be selected depending on the tissue types.
引文
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[15] 莫俊銮, 梁雄顺, 周继昌, 等. 硒干预和DNCB致敏实验小鼠的基因表达分析参考基因的筛选[J]. 中国热带医学, 2016,16(9):861-865.
[16] 李昊文, 刘丽, 郭安臣, 等. 胶质瘤组织实时定量PCR检测中内参基因的选择[J]. 中华临床医师杂志(电子版), 2013,7(8):3384-3388.
[2] AGGARWAL A, JAMWAL M, GANESH K V, et al. Optimal reference gene selection for expression studies in human reticulocytes[J]. J Mol Diagn, 2018,20(3):326-333.
[3] 陈瑞, 杨晓农, 曾婉秋, 等. 家兔不同发育阶段和组织中内参基因的稳定性分析[J]. 畜牧兽医学报, 2016,47(3):477-483.
[4] VEAZEY K J, GOLDING M C. Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells[J]. PLoS One, 2011,6(11):e27592.
[5] SINGH V, KAUL S C, WADHWA R, et al. Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L.) Dunal[J]. PLoS One, 2015,10(3):e118860.
[6] WANG Q, ISHIKAWA T, MICHIUE T, et al. Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper[J]. Int J Legal Med, 2012,126(6):943-952.
[7] XIE F, SUN G, STILLER J W, Et al. Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database[J]. PLoS One, 2011,6(11):e26980.
[8] 王舟, 周继昌, 张慧敏, 等. 不同剂量硒对大鼠四种组织器官硒蓄积浓度的影响[J]. 中国地方病防治杂志, 2014,29(3):165-168.
[9] 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):H34.
[10] ANDERSEN C L, JENSEN J L, ORNTOFT T F. 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 Res, 2004,64(15):5245-5250.
[11] PFAFFL M W, TICHOPAD A, PRGOMET C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeper-excel-based tool using pair-wise correlations[J]. Biotechnol Lett, 2004,26(6):509-515.
[12] SILVER N, BEST S, JIANG J, et al. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR[J]. BMC Mol Biol, 2006,7:33.
[13] DYMOND J S. Explanatory chapter: quantitative PCR[J]. Methods Enzymol, 2013,529:279-289.
[14] CUMMINGS M, SARVESWARAN J, HOMER-VANNIASINKAM S, et al. Glyceraldehyde-3-phosphate dehydrogenase is an inappropriate housekeeping gene for normalising gene expression in sepsis[J]. Inflammation, 2014,37(5):1889-1894.
[15] 莫俊銮, 梁雄顺, 周继昌, 等. 硒干预和DNCB致敏实验小鼠的基因表达分析参考基因的筛选[J]. 中国热带医学, 2016,16(9):861-865.
[16] 李昊文, 刘丽, 郭安臣, 等. 胶质瘤组织实时定量PCR检测中内参基因的选择[J]. 中华临床医师杂志(电子版), 2013,7(8):3384-3388.