mRNA quantification is very important in molecular biological researches. Traditional spectrophotometric method cannot distinguish DNA, rRNA and tRNA species from mRNA. Northern blot can be used for mRNA quantificatio...mRNA quantification is very important in molecular biological researches. Traditional spectrophotometric method cannot distinguish DNA, rRNA and tRNA species from mRNA. Northern blot can be used for mRNA quantification but is known to be time consuming. To rapidly detect mRNA levels, we developed an optical thin-film biosensor chip based method, to quantify mRNA in samples. After total RNA was extracted, the mRNA with poly（A） tails was reverse transcribed with oligo（dT）20 primers and dNTPs mixed with digoxigenin（DIG）-11-dUTE The transcribed first strand cDNA was hybridized with oligo（dA）20 nucleotide probes spotted on optical thin-film biosensor chips. Excess first strand cDNA, single-strand RNA, and mis-matched DNA/DNA hybrids were removed by washing. The perfect-matched DNA/DNA hybrid was detected with anti-DIG-AP （alkaline phosphatase） conjugate and then incubated with NBT/BCIP substrate for color development. The range of the color is from purplish red to blue, according to the cDNA mass deposited on chip sur- face. Detection of mRNA levels from Arabidopsis samples proved that this method is feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.展开更多
Three rice varieties, Zhonghan 3, Shanyou 63 and Aizizhan, were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress. The experiment was pe...Three rice varieties, Zhonghan 3, Shanyou 63 and Aizizhan, were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress. The experiment was performed by gene chip and mRNA differential display technologies under the conditions of drought simulated with 10% PEG6000 solution. The results indicated that the methyl cycle could be activated in the leaves of Zhonghan 3 and Shanyou 63 but inhibited in the leaves of Aizizhan under drought stress. Furthermore, drought stress could induce the expression of a large number of methyltransferase genes, especially the transcription of Rubisco protein methylation related genes, which are beneficial for prevention of Rubisco protein oxidation and degradation, and drought stress could inhibit the transcription of DNA methyltransferase genes and histone methyltransferase genes. This result confirmed that the active methyl cycle and transfer related genes were involved in rice drought resistance.展开更多
目的:考察小牛血去蛋白提取物(DECB)在急性缺氧条件下对小鼠耐缺氧的作用,并初步探索其调节肝脏中能量代谢的可能机制。方法:构建急性缺氧小鼠模型,考察空白组(CON)、模型组(MOD),模型给药组(MOD+DECB)小鼠耐缺氧时间,并检测小鼠肝脏、...目的:考察小牛血去蛋白提取物(DECB)在急性缺氧条件下对小鼠耐缺氧的作用,并初步探索其调节肝脏中能量代谢的可能机制。方法:构建急性缺氧小鼠模型,考察空白组(CON)、模型组(MOD),模型给药组(MOD+DECB)小鼠耐缺氧时间,并检测小鼠肝脏、血清中糖、乳酸、ATP、酮体、三酰甘油(TG)及总胆固醇(TC)的含量,采用mRNA芯片筛选DECB处理后小鼠肝脏中差异表达基因,并进行聚类分析及Real time PCR验证。结果:MOD+DECB组小鼠耐缺氧时间明显高于MOD组,差异具有显著性(P<0.05);MOD+DECB组血清中糖、乳酸含量显著低于MOD组,酮体含量显著高于MOD组,MOD+DECB组肝脏中糖、ATP和酮体的含量显著高于MOD组,乳酸含量显著低于MOD组,差异具有显著性(P<0.01)。肝脏和血清中TG及TC含量各组之间无显著差异。mRNA表达谱芯片分析共筛选出1186个差异表达基因,其中上调表达基因495个,占基因总数的41.7%,下调表达基因691个,占基因总数的58.3%,其中Hmgcs2、Cpt1a、Angptl4、Cyp8b1、Ehhadh基因参与小鼠机体能量代谢密切相关,Real time PCR验证结果与芯片分析结果一致。结论:DECB可通过调控Hmgcs2、Cpt1a、Angptl4、Cyp8b1、Ehhadh基因的表达,调节肝脏中能量的代谢,从而提高小鼠耐缺氧的能力。展开更多
Objective：To study the characteristics of lymphocyte nuclear factor kappa B（NF-κB） signal transduction kinase-related molecular mRNA differential expressions at various month age segments in aging process and the ...Objective：To study the characteristics of lymphocyte nuclear factor kappa B（NF-κB） signal transduction kinase-related molecular mRNA differential expressions at various month age segments in aging process and the intervening effect of Epimedium flavonoids（EF） on it.Methods：Sixty SD rats were divided into six groups,according to animals＇ age,i.e.,the 3 days（d） group,the 4 months（m） group,the 10 m group,the 18 m group,the 27 m group,and the 27 m＋EF group.RNA was extracted from separated splenic lymphocytes. Adopting NF-κB signal path functional genome oligonucleotide gene-chip（128 related genes）,the integral characteristics and differences of NF-κB signal transduction kinase-related mRNA expressions were determined, and the intervening effect of EF was examined.Results：The mean level of the NF-κB signal transduction kinase-related mRNA expressions in rats＇ splenic lymphocytes lowered with aging;the highest expression was presented at 3 d after birth,and then,it lowered gradually,with the lowest level at 18 m or 27 m.After EF intervention,the expression level was raised to the 10-18 m level in the aged rats.Conclusion：The changing rules of lymphocyte NF-κB-signal-transduction-kinase-related mRNA expressions in various stages of aging are helpful for selecting the well time for preventing and intervening aging,and will also give a hint to the molecular index for assessment of senility retarding researches.展开更多
文摘mRNA quantification is very important in molecular biological researches. Traditional spectrophotometric method cannot distinguish DNA, rRNA and tRNA species from mRNA. Northern blot can be used for mRNA quantification but is known to be time consuming. To rapidly detect mRNA levels, we developed an optical thin-film biosensor chip based method, to quantify mRNA in samples. After total RNA was extracted, the mRNA with poly（A） tails was reverse transcribed with oligo（dT）20 primers and dNTPs mixed with digoxigenin（DIG）-11-dUTE The transcribed first strand cDNA was hybridized with oligo（dA）20 nucleotide probes spotted on optical thin-film biosensor chips. Excess first strand cDNA, single-strand RNA, and mis-matched DNA/DNA hybrids were removed by washing. The perfect-matched DNA/DNA hybrid was detected with anti-DIG-AP （alkaline phosphatase） conjugate and then incubated with NBT/BCIP substrate for color development. The range of the color is from purplish red to blue, according to the cDNA mass deposited on chip sur- face. Detection of mRNA levels from Arabidopsis samples proved that this method is feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.
基金supported by the Open Research Fund Program of Jiangsu Key Laboratory of Crop Cultivation and Physiology,China (Grant No.0273880036)
文摘Three rice varieties, Zhonghan 3, Shanyou 63 and Aizizhan, were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress. The experiment was performed by gene chip and mRNA differential display technologies under the conditions of drought simulated with 10% PEG6000 solution. The results indicated that the methyl cycle could be activated in the leaves of Zhonghan 3 and Shanyou 63 but inhibited in the leaves of Aizizhan under drought stress. Furthermore, drought stress could induce the expression of a large number of methyltransferase genes, especially the transcription of Rubisco protein methylation related genes, which are beneficial for prevention of Rubisco protein oxidation and degradation, and drought stress could inhibit the transcription of DNA methyltransferase genes and histone methyltransferase genes. This result confirmed that the active methyl cycle and transfer related genes were involved in rice drought resistance.
文摘目的:考察小牛血去蛋白提取物(DECB)在急性缺氧条件下对小鼠耐缺氧的作用,并初步探索其调节肝脏中能量代谢的可能机制。方法:构建急性缺氧小鼠模型,考察空白组(CON)、模型组(MOD),模型给药组(MOD+DECB)小鼠耐缺氧时间,并检测小鼠肝脏、血清中糖、乳酸、ATP、酮体、三酰甘油(TG)及总胆固醇(TC)的含量,采用mRNA芯片筛选DECB处理后小鼠肝脏中差异表达基因,并进行聚类分析及Real time PCR验证。结果:MOD+DECB组小鼠耐缺氧时间明显高于MOD组,差异具有显著性(P<0.05);MOD+DECB组血清中糖、乳酸含量显著低于MOD组,酮体含量显著高于MOD组,MOD+DECB组肝脏中糖、ATP和酮体的含量显著高于MOD组,乳酸含量显著低于MOD组,差异具有显著性(P<0.01)。肝脏和血清中TG及TC含量各组之间无显著差异。mRNA表达谱芯片分析共筛选出1186个差异表达基因,其中上调表达基因495个,占基因总数的41.7%,下调表达基因691个,占基因总数的58.3%,其中Hmgcs2、Cpt1a、Angptl4、Cyp8b1、Ehhadh基因参与小鼠机体能量代谢密切相关,Real time PCR验证结果与芯片分析结果一致。结论:DECB可通过调控Hmgcs2、Cpt1a、Angptl4、Cyp8b1、Ehhadh基因的表达,调节肝脏中能量的代谢,从而提高小鼠耐缺氧的能力。
基金Supported by the National Natural Science Foundation of China(No.30500681,30973845)Major State Basic Research Development Program of China(No.2007CB507406)Traditional Chinese Medicine Modernization Programs of Shanghai Science and Technology Committee(No.09dZ1975000)
文摘Objective：To study the characteristics of lymphocyte nuclear factor kappa B（NF-κB） signal transduction kinase-related molecular mRNA differential expressions at various month age segments in aging process and the intervening effect of Epimedium flavonoids（EF） on it.Methods：Sixty SD rats were divided into six groups,according to animals＇ age,i.e.,the 3 days（d） group,the 4 months（m） group,the 10 m group,the 18 m group,the 27 m group,and the 27 m＋EF group.RNA was extracted from separated splenic lymphocytes. Adopting NF-κB signal path functional genome oligonucleotide gene-chip（128 related genes）,the integral characteristics and differences of NF-κB signal transduction kinase-related mRNA expressions were determined, and the intervening effect of EF was examined.Results：The mean level of the NF-κB signal transduction kinase-related mRNA expressions in rats＇ splenic lymphocytes lowered with aging;the highest expression was presented at 3 d after birth,and then,it lowered gradually,with the lowest level at 18 m or 27 m.After EF intervention,the expression level was raised to the 10-18 m level in the aged rats.Conclusion：The changing rules of lymphocyte NF-κB-signal-transduction-kinase-related mRNA expressions in various stages of aging are helpful for selecting the well time for preventing and intervening aging,and will also give a hint to the molecular index for assessment of senility retarding researches.
