目的建立real time逆转录聚合酶链反应(RT-PCR)检测BDNF mRNA基因表达的方法.方法提取脑缺血组织的总RNA,进行RT-PCR扩增BDNF mRNA特异性片段,扩增产物重组到质粒上并测序,建立real time RT-PCR检测BDNF mRNA表达水平方法.结果重组的质...目的建立real time逆转录聚合酶链反应(RT-PCR)检测BDNF mRNA基因表达的方法.方法提取脑缺血组织的总RNA,进行RT-PCR扩增BDNF mRNA特异性片段,扩增产物重组到质粒上并测序,建立real time RT-PCR检测BDNF mRNA表达水平方法.结果重组的质粒经酶切和测序,目的片段已插入到载体内,得到real time RT-PCR动力学曲线.结论成功建立real time RT-PCR检测BDNF mRNA基因表达的方法.展开更多
目的:相对定量检测Runx2mRNA在甲状腺乳头状癌和腺瘤中的表达,并探讨其在甲状腺乳头状癌的发生发展及与钙化的意义。方法:相对定量real time RT-PCR检测14例甲状腺乳头状癌和14例甲状腺腺瘤中Runx2mRNA的表达。结果:癌组和腺瘤组的ΔCT...目的:相对定量检测Runx2mRNA在甲状腺乳头状癌和腺瘤中的表达,并探讨其在甲状腺乳头状癌的发生发展及与钙化的意义。方法:相对定量real time RT-PCR检测14例甲状腺乳头状癌和14例甲状腺腺瘤中Runx2mRNA的表达。结果:癌组和腺瘤组的ΔCT值分别为2.395±0.302和5.028±1.179,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01);癌组和腺瘤组的2-ΔΔCT分别为7.826±5.004和1,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01)。癌组和腺瘤组内以钙化分组ΔCT结果示P>0.05,组间差异均无统计学意义。癌组按肿瘤大小<1cm和≥1cm分组,ΔCT值分别为2.629±0.300和2.212±0.124,P<0.05;2-ΔΔCT值分别为167.33±33.823和221.69±18.843,P<0.01。癌组和腺瘤组以及癌组内钙化分组TSH水平比较均P>0.05。结论:Runx2在甲状腺乳头状癌中的表达高,并与癌大小有关,在较大的癌中表达较高。Runx2与微钙化的关系,可能与甲状腺乳头状癌内钙化灶的产生及癌的发生发展有关,在其他的恶性肿瘤(如乳腺癌、前列腺癌、骨肉瘤)也有相关研究。展开更多
猴逆转录病毒(Simian type D retrovirus,SRV)是引起猴获得性免疫缺陷综合征(Simian acquired immunodeficiency syndrome,SAIDS)的病原之一,其严重危害猴的健康,并威胁与猴接触人员的健康,是无特定病原体(SPF)猴必须排除的病毒之一。...猴逆转录病毒(Simian type D retrovirus,SRV)是引起猴获得性免疫缺陷综合征(Simian acquired immunodeficiency syndrome,SAIDS)的病原之一,其严重危害猴的健康,并威胁与猴接触人员的健康,是无特定病原体(SPF)猴必须排除的病毒之一。为了应对口岸对进出境野生及实验用灵长类动物SRV感染情况的监测和流行病学调查的需要,建立了RT-PCR和real-time RT-PCR检测SRV的方法,并对方法的特异性、敏感性和稳定性进行了验证。展开更多
目的 比较Real Time RT-PCR与直接免疫荧光法检测鼠肺中汉坦病毒（hantavirus,HV）带毒率的差异,为进一步完善实验室检测方案提供依据.方法 2015年秋季内蒙古自治区呼伦贝尔市莫力达瓦旗肾综合征出血热（hemorrhagic fever with renal s...目的 比较Real Time RT-PCR与直接免疫荧光法检测鼠肺中汉坦病毒（hantavirus,HV）带毒率的差异,为进一步完善实验室检测方案提供依据.方法 2015年秋季内蒙古自治区呼伦贝尔市莫力达瓦旗肾综合征出血热（hemorrhagic fever with renal syndrome,HFRS）疫区捕获啮齿类动物,对捕获的啮齿类动物进行分类鉴定.取122只鉴定后的鼠进行无菌解剖取肺组织,提取RNA,用Real Time RT-PCR扩增汉坦病毒目的基因,同时将122份肺组织冷冻切片,进行直接免疫荧光试验（direct immunofluorescenee,DIF）.结果 呼伦贝尔市莫力达瓦旗HFRS疫区是以黑线姬鼠为主的混合疫区,捕获鼠类8种共422只,鼠类平均密度为14.81％.在检测的122份鼠肺中,Real Time RT-PCR结果显示：27份鼠肺样品检测到HV核酸阳性,且均为HTNV型,鼠带毒率为22.13％,其中黑线姬鼠16份,占阳性鼠的59.26％.直接免疫荧光法结果为：3份鼠肺样本为阳性,鼠带毒率为2.459％,3份阳性鼠肺均为黑线姬鼠.利用统计软件SPSS 14.0进行统计学分析,两种方法检测结果有统计学差异（x2=21.892,P＜0.01）.结论 与DIF法相比,Real Time RT-PCR检测HV阳性率更高,采用DIF方法可能会低估内蒙古自治区肾综合征出血热疫区的疫情情况.展开更多
Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without val...Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.展开更多
文摘目的建立real time逆转录聚合酶链反应(RT-PCR)检测BDNF mRNA基因表达的方法.方法提取脑缺血组织的总RNA,进行RT-PCR扩增BDNF mRNA特异性片段,扩增产物重组到质粒上并测序,建立real time RT-PCR检测BDNF mRNA表达水平方法.结果重组的质粒经酶切和测序,目的片段已插入到载体内,得到real time RT-PCR动力学曲线.结论成功建立real time RT-PCR检测BDNF mRNA基因表达的方法.
文摘目的:相对定量检测Runx2mRNA在甲状腺乳头状癌和腺瘤中的表达,并探讨其在甲状腺乳头状癌的发生发展及与钙化的意义。方法:相对定量real time RT-PCR检测14例甲状腺乳头状癌和14例甲状腺腺瘤中Runx2mRNA的表达。结果:癌组和腺瘤组的ΔCT值分别为2.395±0.302和5.028±1.179,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01);癌组和腺瘤组的2-ΔΔCT分别为7.826±5.004和1,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01)。癌组和腺瘤组内以钙化分组ΔCT结果示P>0.05,组间差异均无统计学意义。癌组按肿瘤大小<1cm和≥1cm分组,ΔCT值分别为2.629±0.300和2.212±0.124,P<0.05;2-ΔΔCT值分别为167.33±33.823和221.69±18.843,P<0.01。癌组和腺瘤组以及癌组内钙化分组TSH水平比较均P>0.05。结论:Runx2在甲状腺乳头状癌中的表达高,并与癌大小有关,在较大的癌中表达较高。Runx2与微钙化的关系,可能与甲状腺乳头状癌内钙化灶的产生及癌的发生发展有关,在其他的恶性肿瘤(如乳腺癌、前列腺癌、骨肉瘤)也有相关研究。
文摘猴逆转录病毒(Simian type D retrovirus,SRV)是引起猴获得性免疫缺陷综合征(Simian acquired immunodeficiency syndrome,SAIDS)的病原之一,其严重危害猴的健康,并威胁与猴接触人员的健康,是无特定病原体(SPF)猴必须排除的病毒之一。为了应对口岸对进出境野生及实验用灵长类动物SRV感染情况的监测和流行病学调查的需要,建立了RT-PCR和real-time RT-PCR检测SRV的方法,并对方法的特异性、敏感性和稳定性进行了验证。
文摘目的 比较Real Time RT-PCR与直接免疫荧光法检测鼠肺中汉坦病毒（hantavirus,HV）带毒率的差异,为进一步完善实验室检测方案提供依据.方法 2015年秋季内蒙古自治区呼伦贝尔市莫力达瓦旗肾综合征出血热（hemorrhagic fever with renal syndrome,HFRS）疫区捕获啮齿类动物,对捕获的啮齿类动物进行分类鉴定.取122只鉴定后的鼠进行无菌解剖取肺组织,提取RNA,用Real Time RT-PCR扩增汉坦病毒目的基因,同时将122份肺组织冷冻切片,进行直接免疫荧光试验（direct immunofluorescenee,DIF）.结果 呼伦贝尔市莫力达瓦旗HFRS疫区是以黑线姬鼠为主的混合疫区,捕获鼠类8种共422只,鼠类平均密度为14.81％.在检测的122份鼠肺中,Real Time RT-PCR结果显示：27份鼠肺样品检测到HV核酸阳性,且均为HTNV型,鼠带毒率为22.13％,其中黑线姬鼠16份,占阳性鼠的59.26％.直接免疫荧光法结果为：3份鼠肺样本为阳性,鼠带毒率为2.459％,3份阳性鼠肺均为黑线姬鼠.利用统计软件SPSS 14.0进行统计学分析,两种方法检测结果有统计学差异（x2=21.892,P＜0.01）.结论 与DIF法相比,Real Time RT-PCR检测HV阳性率更高,采用DIF方法可能会低估内蒙古自治区肾综合征出血热疫区的疫情情况.
基金Supported by the Knowledge Innovation Program of Chinese Academy of Sciences(No.KSCX2-EW-G-12B)the Knowledge Innovation Program of the Chinese Academy of Sciences(No.KZCX2-EW-Q213)the National High Technology Research and Development Program of China (863 Program)(No.2012AA10A412)
文摘Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.
