An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombi...An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.展开更多
In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samp...In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.展开更多
目的：基于双链嵌合荧光染料SYBR Green Ⅰ，建立一种快速、灵敏的检测大鼠Ⅰ型胶原mRNA表达水平的实时逆转录聚合酶链反应（RT-PCR）方法．方法：提取大鼠肝脏总RNA，RT-PCR扩增Ⅰ型胶原基因部分片段，将其克隆入pMD18-T载体用作参比...目的：基于双链嵌合荧光染料SYBR Green Ⅰ，建立一种快速、灵敏的检测大鼠Ⅰ型胶原mRNA表达水平的实时逆转录聚合酶链反应（RT-PCR）方法．方法：提取大鼠肝脏总RNA，RT-PCR扩增Ⅰ型胶原基因部分片段，将其克隆入pMD18-T载体用作参比品．基于SYBR GreenⅠ双链嵌合染料建立检测大鼠Ⅰ型胶原mRNA表达水平方法．其中对一系列连续稀释的参比品进行实时PCR分析，评价所建立方法的检测灵敏度；对PCR产物的熔解曲线进行分析评价其特异性．结果：重组质粒构建成功，经测序鉴定，目的片段已插入pMD18-T载体内．所建立的实时RT-PCR方法的最低检测限度为1个拷贝／反应，在每反应10^0～10^7拷贝范围内，CT值（C代表cycle，T代表threshold；CT值指荧光信号达到设定的阈值所经历的循环数）与起始模板浓度具有良好的线性关系，相关系数为0．991．结论：所建立的基于SYBR GreenⅠ双链嵌合染料的大鼠Ⅰ型胶原PCR检测方法具有敏感性高、特异性强和线性检测范围广等特点，适用于进一步的各种组织的大量样本检测．展开更多
文摘An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.
文摘In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.
文摘目的：基于双链嵌合荧光染料SYBR Green Ⅰ，建立一种快速、灵敏的检测大鼠Ⅰ型胶原mRNA表达水平的实时逆转录聚合酶链反应（RT-PCR）方法．方法：提取大鼠肝脏总RNA，RT-PCR扩增Ⅰ型胶原基因部分片段，将其克隆入pMD18-T载体用作参比品．基于SYBR GreenⅠ双链嵌合染料建立检测大鼠Ⅰ型胶原mRNA表达水平方法．其中对一系列连续稀释的参比品进行实时PCR分析，评价所建立方法的检测灵敏度；对PCR产物的熔解曲线进行分析评价其特异性．结果：重组质粒构建成功，经测序鉴定，目的片段已插入pMD18-T载体内．所建立的实时RT-PCR方法的最低检测限度为1个拷贝／反应，在每反应10^0～10^7拷贝范围内，CT值（C代表cycle，T代表threshold；CT值指荧光信号达到设定的阈值所经历的循环数）与起始模板浓度具有良好的线性关系，相关系数为0．991．结论：所建立的基于SYBR GreenⅠ双链嵌合染料的大鼠Ⅰ型胶原PCR检测方法具有敏感性高、特异性强和线性检测范围广等特点，适用于进一步的各种组织的大量样本检测．
