以石蒜属植物换锦花(Lycoris sprengeri)、石蒜(Lycoris radiata)和中国石蒜(Lycoris chinensis Traub)不同组织器官、不同花发育时期以及不同杂交种的幼小鳞茎为研究材料,利用q RT-PCR技术检测Actin、EF-1α、GAPDH、5S r RNA、Ubiqui...以石蒜属植物换锦花(Lycoris sprengeri)、石蒜(Lycoris radiata)和中国石蒜(Lycoris chinensis Traub)不同组织器官、不同花发育时期以及不同杂交种的幼小鳞茎为研究材料,利用q RT-PCR技术检测Actin、EF-1α、GAPDH、5S r RNA、Ubiquitin和β-Tubulin等6个内参基因的m RNA表达情况,利用ge Norm、Norm Finder、Best Keeper和Reffinder软件综合评价6个内参基因的表达稳定性。结果表明,石蒜属植物种间和种内内参基因表达差异明显,在分析换锦花不同组织基因表达时可选用β-Tubulin、Actin和GAPDH作为内参基因,分析不同花期的基因表达时宜选用5S r RNA、EF-1α、Actin和β-Tubulin作内参基因。中国石蒜不同组织最合适的内参基因是5S r RNA、Ubiquitin、EF-1α和β-Tubulin,而不同花期最合适的内参基因则是Ubiquitin、β-Tubulin。分析石蒜不同组织间基因表达的适宜内参基因是β-Tubulin和5S r RNA,不同花期为Ubiquitin、β-Tubulin以及5S r RNA。不同杂交种鳞茎适宜内参基因为β-Tubulin和Actin。展开更多
目的建立鲜草莓中GⅡ型诺如病毒(No V GⅡ)的实时荧光RT-PCR检测方法,评价磁珠富集法和PEG(聚乙二醇)沉淀法对检测草莓中No V GⅡ的适用性,对北京地区采集的18份草莓样品进行检测。方法参照ISO/TS 15216-1《实时荧光RT-PCR方法测定食品...目的建立鲜草莓中GⅡ型诺如病毒(No V GⅡ)的实时荧光RT-PCR检测方法,评价磁珠富集法和PEG(聚乙二醇)沉淀法对检测草莓中No V GⅡ的适用性,对北京地区采集的18份草莓样品进行检测。方法参照ISO/TS 15216-1《实时荧光RT-PCR方法测定食品中甲型肝炎病毒和诺如病毒水平方法》合成检测No V GⅡ的特异性引物和探针,分别采用磁珠富集法和PEG沉淀法富集病毒,然后提取RNA,建立实时荧光RT-PCR检测方法,并对提取条件进行优化。结果磁珠富集法的最高回收率为1.730%(PBS缓冲液),PEG沉淀法的最高回收率为1.682%(TGBE缓冲液),18份草莓样品均未检出No V GⅡ。结论通过磁珠富集法和PEG沉淀法建立的病毒检测的富集方法均适用于鲜草莓中No V GⅡ的检测。展开更多
BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and ser...BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and sera and its gene transcription for diagnosis and monitoring of metastasis of HCC. METHODS: Liver GPC-3 was analyzed in HCC tissues from 36 patients by immunohistochemistry and Western blotting. GPC-3 mRNA from circulating peripheral blood mononuclear cells from 123 HCC patients or 246 patients with other diseases or 36 HCC tissues was amplified by RT-PCR, quantitative realtime PCR, and confirmed by DNA sequencing. Circulating GPC-3 level was detected by ELISA. RESULTS: The increasing expression of GPC-3 was observed from non-cancerous to cancerous tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. The positive rate of GPC-3 was 80.6% in HCC, 41.7% in their paracancerous tissues, and none in distal cancerous tissues (P【0.001), with no significant difference in differentiation grade and tumor number except for size (Z=2.941, P=0.003). Serum GPC-3 was detected only in HCC (52.8%) and significant difference was found between GPC-3 and tumor size (χ2 =6.318, P=0.012) or HBV infection (χ2 =23.362, P【0.001). Circulating GPC-3 mRNA was detected in 70.7% of HCC tissues, with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis (P【0.001). The combination ofcirculating GPC-3, GPC-3 mRNA and alpha-fetoprotein is of complementary value for HCC diagnosis (94.3%). CONCLUSION: Both GPC-3 overexpression and GPC-3 mRNA abnormality could be used as markers for the diagnosis of HCC and monitoring its metastasis.展开更多
文摘以石蒜属植物换锦花(Lycoris sprengeri)、石蒜(Lycoris radiata)和中国石蒜(Lycoris chinensis Traub)不同组织器官、不同花发育时期以及不同杂交种的幼小鳞茎为研究材料,利用q RT-PCR技术检测Actin、EF-1α、GAPDH、5S r RNA、Ubiquitin和β-Tubulin等6个内参基因的m RNA表达情况,利用ge Norm、Norm Finder、Best Keeper和Reffinder软件综合评价6个内参基因的表达稳定性。结果表明,石蒜属植物种间和种内内参基因表达差异明显,在分析换锦花不同组织基因表达时可选用β-Tubulin、Actin和GAPDH作为内参基因,分析不同花期的基因表达时宜选用5S r RNA、EF-1α、Actin和β-Tubulin作内参基因。中国石蒜不同组织最合适的内参基因是5S r RNA、Ubiquitin、EF-1α和β-Tubulin,而不同花期最合适的内参基因则是Ubiquitin、β-Tubulin。分析石蒜不同组织间基因表达的适宜内参基因是β-Tubulin和5S r RNA,不同花期为Ubiquitin、β-Tubulin以及5S r RNA。不同杂交种鳞茎适宜内参基因为β-Tubulin和Actin。
文摘目的建立鲜草莓中GⅡ型诺如病毒(No V GⅡ)的实时荧光RT-PCR检测方法,评价磁珠富集法和PEG(聚乙二醇)沉淀法对检测草莓中No V GⅡ的适用性,对北京地区采集的18份草莓样品进行检测。方法参照ISO/TS 15216-1《实时荧光RT-PCR方法测定食品中甲型肝炎病毒和诺如病毒水平方法》合成检测No V GⅡ的特异性引物和探针,分别采用磁珠富集法和PEG沉淀法富集病毒,然后提取RNA,建立实时荧光RT-PCR检测方法,并对提取条件进行优化。结果磁珠富集法的最高回收率为1.730%(PBS缓冲液),PEG沉淀法的最高回收率为1.682%(TGBE缓冲液),18份草莓样品均未检出No V GⅡ。结论通过磁珠富集法和PEG沉淀法建立的病毒检测的富集方法均适用于鲜草莓中No V GⅡ的检测。
基金supported in part by grants-in-Aid from the Projects of Jiangsu Medical Science (HK201102, H200925)the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD)the Program of Nantong Society Undertaking and Technological Innovation (HS2011012),China
文摘BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and sera and its gene transcription for diagnosis and monitoring of metastasis of HCC. METHODS: Liver GPC-3 was analyzed in HCC tissues from 36 patients by immunohistochemistry and Western blotting. GPC-3 mRNA from circulating peripheral blood mononuclear cells from 123 HCC patients or 246 patients with other diseases or 36 HCC tissues was amplified by RT-PCR, quantitative realtime PCR, and confirmed by DNA sequencing. Circulating GPC-3 level was detected by ELISA. RESULTS: The increasing expression of GPC-3 was observed from non-cancerous to cancerous tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. The positive rate of GPC-3 was 80.6% in HCC, 41.7% in their paracancerous tissues, and none in distal cancerous tissues (P【0.001), with no significant difference in differentiation grade and tumor number except for size (Z=2.941, P=0.003). Serum GPC-3 was detected only in HCC (52.8%) and significant difference was found between GPC-3 and tumor size (χ2 =6.318, P=0.012) or HBV infection (χ2 =23.362, P【0.001). Circulating GPC-3 mRNA was detected in 70.7% of HCC tissues, with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis (P【0.001). The combination ofcirculating GPC-3, GPC-3 mRNA and alpha-fetoprotein is of complementary value for HCC diagnosis (94.3%). CONCLUSION: Both GPC-3 overexpression and GPC-3 mRNA abnormality could be used as markers for the diagnosis of HCC and monitoring its metastasis.
