摘要
分析取材前不同果糖喂养(禁食给清水和禁食给果糖水)条件下,大鼠肝脏常用内参基因m RNA的稳定性,筛选出最适宜的内参,以保证实时荧光定量PCR(real-time PCR)结果的可靠性。长期给予10%果糖水建立非酒精性脂肪肝大鼠模型,在动物体重与饮用果糖量相等的情况下,取材前随机分成两组:禁食给清水组(Fru 1);禁食继续给果糖水组(Fru 2);正常对照组(Con)自由饮清水。提取肝脏样本RNA,取等量RNA逆转录合成c DNA,real-time PCR检测18S、GAPDH、ACTB和TBP含量变化,分别利用ge Norm程序和Bio-Rad CFX Manager软件分析这几个基因的稳定性。ge Norm分析得到的稳定性排序为:ACTB>GAPDH>TBP>18S。Bio-Rad CFX Manager软件稳定性排序为:ACTB>TBP>GAPDH>18S。两个软件均得到ACTB最为稳定,TBP次之。然而经3次重复逆转录基因表达分析,相对于Con组和Fru1组,Fru 2组ACTB与GAPDH明显升高,18S略有下降,TBP在3组间比较稳定。结合肝脏脂质合成相关的两个关键基因Ch REBP,SREBP1c表达变化分析,分别以这4种基因为内参计算这两个基因在3组间的含量变化。只有以GAPDH为内参时,Fru 2组Ch REBP表达量相对于Fru 1没有明显上升,其它的表达趋势一致,只是含量高低有所区别。因此,我们认为TBP和ACTB均可作为不同果糖喂养状态下大鼠肝脏的内参基因。
Expression stability of candidate reference genes （18S, GA PDH, A CTB, TBP） was tested for quantitative real-time PCR, to select a suitable reference gene for qRT-PCR normalization in rats induced by different fructose feeding （fasting and the fructose solution was replaced with tap water; fasting and continued supplementation with the fructose solution）. SD rats were treated with 10% fructose water for long-term to establish non-alcoholic fatty liver model. Under the condition that their weights and fructose content were similar, they were divided into the following two groups before sacrifice： half of the rats （group of Fru 1） were deprived fructose solution by tap water, and the other （group of Fru 2） were still treated with 10% fructose. Normal rats with unlimited water were severed as control group, and all rats were under chow-fasting. RNAs were extracted from the liver samples and cDNA was synthesized. Reference genes 18S, GAPDH, A CTB and TBP in liver were tested by Real-time PCR, their stability were respectively analysized by geNorm and Bio-Rad CFX Manager software. The expression stability of four genes was （from high to low）： A CTB〉GA PDH〉TBP〉18S by geNorm. The expression stability of four genes was A CTB〉 TBP〉GAPDH〉18S by Bio-Rad CFX Manager soRware. Two softwares both indicated ACTB was stablest and TBP was the second stablest. However, according to three expression analysis of reverse transcription gene, A CTB and GA PDH expressions in group of Fru 2 were significantly higher than those in group of Con and group of Fru 1. In Fru 2, the expression of 18S decreased while TBP showed the stable level in three groups. Further, expressions of hepatic prominent lipogenic genes ChREBP and SREBPlc were rated to four interior reference genes respectively to further assess their stability. ChREBP expression in group of Fru 2 did not elevate obviously compared with those in group of Con and group of Frul when GA PDH severed as interior reference. Others expression trends were consistent, but the levels were different. Our result suggested that A CTB and TBP gene might be the stable refer- ence genes for liver of rats under different fructose-feeding conditions.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2017年第8期3315-3321,共7页
Genomics and Applied Biology
基金
国家自然科学基金(81374033
81673659)
重庆市教委课题(KJ1600233)
重庆市渝中区科委课题(20120220)
重庆医科大学培育基金(X12100)共同资助
