| 奶牛乳腺上皮细胞HSP70 mRNA荧光实时定量PCR标准曲线的建立 |
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| 引用本文： | 赵丽红,高艳霞,李秋凤,李建国. 奶牛乳腺上皮细胞HSP70 mRNA荧光实时定量PCR标准曲线的建立[J]. 河北农业大学学报, 2010, 33(5) |
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| 作者姓名： | 赵丽红 高艳霞 李秋凤 李建国 |
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| 作者单位： | 河北农业大学,动物科技学院,河北保定,071001;河北农业大学,动物科技学院,河北保定,071001;河北农业大学,动物科技学院,河北保定,071001;河北农业大学,动物科技学院,河北保定,071001 |
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| 基金项目： | 现代农业产业技术体系建设专项资金资助 |
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| 摘 要： | 为构建奶牛乳腺上皮细胞HSP70 mRNA表达水平差异的标准品,以体外培养的奶牛乳腺上皮细胞总RNA为模板,反转录成cDNA,用该cDNA为模板进行PCR,扩增目的基因片段,构建pGEM-T-HSP70重组质粒,鉴定测序后,用荧光PCR构建标准曲线。结果构建的HSP70质粒经PCR扩增后,得到了明显的259 bp条带,测序结果与目的条带完全一致,且质粒原始浓度为2.5×1010copies/mL,倍比稀释至2.5×103copies/mL,均能得到良好的标准曲线(R=0.99),提示构建的HSP70标准质粒成功。
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| 关 键 词： | HSP70 荧光实时定量PCR 质粒 |
The HSP70 mRNA of mammary epithelial cells by real time FQ-PCR standard plasmid preparation and standard curve establishment |
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| ZHAO Li-hong,GAO Yan-xia,LI Qiu-feng,LI Jian-guo. The HSP70 mRNA of mammary epithelial cells by real time FQ-PCR standard plasmid preparation and standard curve establishment[J]. Journal of Agricultural University of Hebei, 2010, 33(5) |
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| Authors: | ZHAO Li-hong GAO Yan-xia LI Qiu-feng LI Jian-guo |
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| Affiliation: | ZHAO Li-hong,GAO Yan-xia,LI Qiu-feng,LI Jian-guo(College of Animal Science , Technology,Agricultural University of Hebei,Baoding 071000,China) |
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| Abstract: | To clone dairy cow HSP70 cDNA and use it as the standard for real-time quantifying HSP70 mRNA.Total RNA was extracted from vitro raised cow mammary gland epithelial cells and reverse transcribed to cDNA.Ampified HSP70 and identified with polymerase clain reaction(PCR),and then were subjected to sequencing.Total RNA extracted from dairy caw mammary gland epithelial cells were perfected.The PCR of HSP70 plasmid was a 259 bp clear stripe,and the DNA sequencing of HSP70 was according with aimed fragment(100%).T... |
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| Keywords: | HSP70 |
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