摘要
研究旨在提高miRNA模拟物在奶牛子宫内膜原代上皮细胞的转染效率。应用带有FAM标记的miRNA-185 mimics为报告基因,检测两种不同转染试剂(Lipofectamine RNAiMAX与Lipofectamine 2000)在细胞接种12、18、24 h后的转染效率,采用Q-PCR法检测最佳转染条件下试验组与对照组miRNA-185的表达变化。结果显示,无论使用Lipofectamine 2000还是Lipofectamine RNAiMAX作为奶牛子宫内膜原代上皮细胞的转染试剂,各组间12 h的转染效率均极显著高于18、24 h(P<0.01),18 h的转染效率均极显著高于24 h(P<0.01);Lipofectamine RNAiMAX各时间点的转染效率均极显著高于Lipofectamine 2000(P<0.01)。使用Lipofectamine RNAiMAX作为奶牛子宫内膜原代上皮细胞的转染试剂时,12 h的荧光强度极显著高于18、24 h(P<0.01),24 h的荧光强度极显著高于18 h(P<0.01),Lipofectamine RNAiMAX各时间点的荧光强度均极显著高于Lipofectamine 2000(P<0.01)。检测Lipofectamine RNAiMAX在细胞接种12 h转染后miRNA-185表达量,发现与对照组相比,试验组miRNA-185的表达极显著升高(P<0.01)。研究表明,奶牛子宫内膜原代上皮细胞适合应用脂质体进行miRNA化学合成模拟物的转染,当选用Lipofectamine RNAiMAX作转染试剂,细胞接种12 h后转染可达最佳的转染效果。
The research aimed to improve the transfection efficiency of miRNA mimics in primary cow endometrial epithelial cells.Using FAM-labeled miRNA-185 mimics as reporter genes,the transfection efficiencies of two different transfection reagents(Lipofectamine RNAiMAX and Lipofectamine 2000)were detected 12,18 and 24 h after cell inoculation.The expression changes of miRNA-185 in the experimental group and the control group were detected by QPCR method under the optimal transfection conditions.The results showed that whether using Lipofectamine 2000 or Lipofectamine RNAiMAX as a transfection reagent for primary cow endometrial epithelial cells,the transfection efficiency of 12 h in each group was extremely higher than that of 18 h and 24 h(P<0.01),and the transfection efficiency of 18 h was extremely higher than that of 24 h(P<0.01).The transfection efficiency of Lipofectamine RNAiMAX at each time point was extremely higher than that of Lipofectamine 2000(P<0.01).When Lipofectamine RNAiMAX was used as the transfection reagent for primary cow endometrial epithelial cells,the fluorescence intensity at 12 h was extremely higher than that at 18 and 24 h(P<0.01),the fluorescence intensity at 24 h was extremely higher than that at 18 h(P<0.01).The fluorescence intensity of Lipofectamine RNAiMAX at each time point was extremely higher than that of Lipofectamine 2000(P<0.01).The expression of miRNA-185 in Lipofectamine RNAiMAX was detected 12 h after cell inoculation,and it was found that compared with the control group,the expression of miRNA185 in the experimental group was extremely increased(P<0.01).The study indicates that the best transfection condition is that Lipofectamine RNAiMAX was used at 12 h in uterine epithelial primary cells of dairy cows.
作者
邹晓
郑程远
孙敬明
张晓山
王平
Zou Xiao;Zheng Chengyuan;Sun Jingming;Zhang Xiaoshan;Wang Ping(Qiqihar University,Heilongjiang Qiqihar 161000;Heilongjiang Beidacang Group Co.,Ltd.,Heilongjiang Qiqihar 161000)
出处
《现代畜牧兽医》
2022年第4期27-31,共5页
Modern Journal of Animal Husbandry and Veterinary Medicine
基金
2018年度黑龙江省省属高等学校基本科研业务费科研项目(135309367)。
