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
The complicated life cycle ofAurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individ...The complicated life cycle ofAurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individuals, such as planulae. To solve this problem, we developed a real-time PCR assay （SYBR Green I） to identify planulae in both cultured and natural seawater samples. Species-specific primers targeting Aurelia sp.1 mitochondrial 16S rDNA （mr 16S rDNA） regions were designed. Using a calibration curve constructed with plasmids containing the Aurelia sp. 1 mt 16S rDNA fragment and a standard curve for planulae, the absolute number of mt 16S rDNA copies per planula was determined and from that the total number ofplanulae per sample was estimated. For the field samples, a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 μg/μL BSA in the PCR reaction mixture was used to remove real- time PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak Aurelia sp.1 planula abundance occurred in July 2012 at stations near Hongdao Island and Qingdao offshore; abundances were very low in August and September. The real-time PCR assay （SYBR Green I） developed here negates the need for traditional microscopic identification, which is laborious and time-consuming, and can detect and quantify jellyfish planulae in field plankton samples rapidly and specifically.展开更多
[Objective] This study aimed to establish a quantitative real-time PCR （qRT-PCR） system for detecting the expression of rice beta-glucosidase gene Os1bglu4.[Method] The PCR was conducted with SYBR Green Ⅰ method,us...[Objective] This study aimed to establish a quantitative real-time PCR （qRT-PCR） system for detecting the expression of rice beta-glucosidase gene Os1bglu4.[Method] The PCR was conducted with SYBR Green Ⅰ method,using the primers of reference gene actin or ubiquitin.[Result] Actin was more suitable to be the reference gene than ubiquitin.More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results,so,0.4 μmol/L was selected as the optimal primer concentration in this study.The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃,therefore,annealing temperature was set at 60 ℃.Compared with the reaction system of 25 μl,the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So,the 10 μl reaction system was selected,which significantly reduces the research costs for the detection of a large amount of samples in future study.展开更多
Encoded by seven genes, angiopoietin-like （ANGPTL） family members structurally similar to the angiogenic regulating factor angiopoietin are known to possess biological activities in angiogenesis and metabolism. Here...Encoded by seven genes, angiopoietin-like （ANGPTL） family members structurally similar to the angiogenic regulating factor angiopoietin are known to possess biological activities in angiogenesis and metabolism. Here we reports for the first time the identification and expression analysis of all the seven members of bovine ANGPTL gene family, which were designated bANGPTL1 to bANGPTL7 in order. The seven bANGPTL genes consist of 4-9 exons, span 3800M-3000 bp and are located on different chromosomes. The deduced amino acid sequences of the members all possess an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain, both characteristics of angiopoietins. Phylogenetic analysis showed that the 32 identified ANGPTL homologs from 9 species could be classified into two major groups. Real-time quantitative PCR （Q-PCR） analysis revealed that the bANGPTL family members have different expression patterns. This study will be helpful for investigation on the biological role of the bANGPTL family in this economically important species. Furthermore, it provides an insight into the molecular evolution of the emerging ANGPTL family展开更多
Objective： The purpose of this paper was to study the expression levels of newly described lymphatic endothelial markers – LYVE-1, Prox-1, podoplanin and 5’-nucleotidase, and their correlation with metastasis of hu...Objective： The purpose of this paper was to study the expression levels of newly described lymphatic endothelial markers – LYVE-1, Prox-1, podoplanin and 5’-nucleotidase, and their correlation with metastasis of human colorectal cancers. Methods： Tumor and corresponding tumor-side normal tissue samples were obtained from resected specimens immediately after operation. Expression level of each factor was determined by quantitative real-time PCR （RT-PCR） and Western blot technique. Results： Expression levels of lymphatic endothelial markers LYVE-1, Prox-1, podoplanin and 5’-nucleotidase were significantly different in tumor and tumor-side normal groups. Expression levels of Prox-1 and podoplanin were higher in patients with positive lymph node metastasis than those without metastasis. LYVE-1, but not 5’-nucleotidase expression level was higher in both cancer and normal groups. Conclusion： These results indicate that combined quantitative analysis of lymphangiogenic markers LYVE-1, Prox-1 and podoplanin in colorectal cancer specimens may be useful in predicting metastasis of colorectal cancer to regional lymph nodes. However, the role of 5’-nucleotidase in predicting metastasis of colorectal cancer still remains to be further analyzed.展开更多
Objective： The aim of the study was to observe the expressions of genes related to genome stability and DNA repair in the members of nasopharyngeal carcinoma （NPC） clustedng families. Methods： In the Zhongshan Cit...Objective： The aim of the study was to observe the expressions of genes related to genome stability and DNA repair in the members of nasopharyngeal carcinoma （NPC） clustedng families. Methods： In the Zhongshan City where there is highly incidence rate of NPC, we chose the members of the NPC clustering families as objects, and the patients of nasopharyngitis and NPC as the control group. We isolated the RNA from the nasopharyngeal tissue, and synthesized its cRNA, the genome stability and DNA repair genes chip technique, chemiluminescent detection and real-time fluorescence quantita- tive technique were used to examine the genome stability and DNA repair genes in the nasopharyngeal tissue. Results： More genome stability and DNA repair genes were up-regulated in the members of the NPC clustering families than the NPC patients, and the range of up-regulated was high, with the over up-regulated 100 times genes including TEP1, MSH4, PMS2LI. Fewer genome stability and DNA repair genes were down-regulated in the members of the NPC clustering families than the NPC patients, the ubiquitin genes almost were down-regulated, the results also could be confirmed by real-time fluorescence quantitative PCR. Conclusion： There are specially expression character of genome stability and DNA repair genes in the members of NPC clustering families.展开更多
The transformer-2(tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster.In this study,sequences and expression profiles of...The transformer-2(tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster.In this study,sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized.Four tra-2 isoforms,designated as Estra-2a,Estra-2b,Estra-2c,and Estra-2d,were isolated.They all contained an RNA-recognition motif(RRM) and a linker region,which shared high similarity with other reported tra-2s.Sequence analysis revealed that Estra-2a,Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA.Compared with the other three isoforms,Estra-2d lacks the RS2 domain.Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg,and in the 2-4 cell and blastula stages compared with larval stages(P<0.01),suggesting their maternal origin in early embryonic developmental stages.Notably,Estra-2a was highly expressed in male somatic tissues,while Estra-2c was significantly highly expressed in the ovary.These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab.Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.展开更多
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.
基金Supported by the National Basic Research Program of China(973 Program)(No.2011CB403602)the National Natural Science Foundation of China(No.41076085)the National Special Research Fund for Non-Profit Marine Sector(No.201205031)
文摘The complicated life cycle ofAurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individuals, such as planulae. To solve this problem, we developed a real-time PCR assay （SYBR Green I） to identify planulae in both cultured and natural seawater samples. Species-specific primers targeting Aurelia sp.1 mitochondrial 16S rDNA （mr 16S rDNA） regions were designed. Using a calibration curve constructed with plasmids containing the Aurelia sp. 1 mt 16S rDNA fragment and a standard curve for planulae, the absolute number of mt 16S rDNA copies per planula was determined and from that the total number ofplanulae per sample was estimated. For the field samples, a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 μg/μL BSA in the PCR reaction mixture was used to remove real- time PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak Aurelia sp.1 planula abundance occurred in July 2012 at stations near Hongdao Island and Qingdao offshore; abundances were very low in August and September. The real-time PCR assay （SYBR Green I） developed here negates the need for traditional microscopic identification, which is laborious and time-consuming, and can detect and quantify jellyfish planulae in field plankton samples rapidly and specifically.
基金Supported by Guizhou International Cooperation Project on Science and Technology[(2013)7040]the 20th Project of the Joint Committee on Scientific and Technical Cooperation between the Government of the Kingdom of Thailand and the Government of the People’s Republic of China (20-606J)the Fund from Suranaree University of Technology,Thailand (SUT3-304-54-12-29)
文摘[Objective] This study aimed to establish a quantitative real-time PCR （qRT-PCR） system for detecting the expression of rice beta-glucosidase gene Os1bglu4.[Method] The PCR was conducted with SYBR Green Ⅰ method,using the primers of reference gene actin or ubiquitin.[Result] Actin was more suitable to be the reference gene than ubiquitin.More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results,so,0.4 μmol/L was selected as the optimal primer concentration in this study.The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃,therefore,annealing temperature was set at 60 ℃.Compared with the reaction system of 25 μl,the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So,the 10 μl reaction system was selected,which significantly reduces the research costs for the detection of a large amount of samples in future study.
基金supported by the Fundamental Research Funds for the Central Universities (Grant No. JUSRP10916)the Program of 'Qinglan Project' of Jiangsu Province
文摘Encoded by seven genes, angiopoietin-like （ANGPTL） family members structurally similar to the angiogenic regulating factor angiopoietin are known to possess biological activities in angiogenesis and metabolism. Here we reports for the first time the identification and expression analysis of all the seven members of bovine ANGPTL gene family, which were designated bANGPTL1 to bANGPTL7 in order. The seven bANGPTL genes consist of 4-9 exons, span 3800M-3000 bp and are located on different chromosomes. The deduced amino acid sequences of the members all possess an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain, both characteristics of angiopoietins. Phylogenetic analysis showed that the 32 identified ANGPTL homologs from 9 species could be classified into two major groups. Real-time quantitative PCR （Q-PCR） analysis revealed that the bANGPTL family members have different expression patterns. This study will be helpful for investigation on the biological role of the bANGPTL family in this economically important species. Furthermore, it provides an insight into the molecular evolution of the emerging ANGPTL family
文摘Objective： The purpose of this paper was to study the expression levels of newly described lymphatic endothelial markers – LYVE-1, Prox-1, podoplanin and 5’-nucleotidase, and their correlation with metastasis of human colorectal cancers. Methods： Tumor and corresponding tumor-side normal tissue samples were obtained from resected specimens immediately after operation. Expression level of each factor was determined by quantitative real-time PCR （RT-PCR） and Western blot technique. Results： Expression levels of lymphatic endothelial markers LYVE-1, Prox-1, podoplanin and 5’-nucleotidase were significantly different in tumor and tumor-side normal groups. Expression levels of Prox-1 and podoplanin were higher in patients with positive lymph node metastasis than those without metastasis. LYVE-1, but not 5’-nucleotidase expression level was higher in both cancer and normal groups. Conclusion： These results indicate that combined quantitative analysis of lymphangiogenic markers LYVE-1, Prox-1 and podoplanin in colorectal cancer specimens may be useful in predicting metastasis of colorectal cancer to regional lymph nodes. However, the role of 5’-nucleotidase in predicting metastasis of colorectal cancer still remains to be further analyzed.
基金Supported by a grant from the National Natural Science Foundation of China (No.30672713)
文摘Objective： The aim of the study was to observe the expressions of genes related to genome stability and DNA repair in the members of nasopharyngeal carcinoma （NPC） clustedng families. Methods： In the Zhongshan City where there is highly incidence rate of NPC, we chose the members of the NPC clustering families as objects, and the patients of nasopharyngitis and NPC as the control group. We isolated the RNA from the nasopharyngeal tissue, and synthesized its cRNA, the genome stability and DNA repair genes chip technique, chemiluminescent detection and real-time fluorescence quantita- tive technique were used to examine the genome stability and DNA repair genes in the nasopharyngeal tissue. Results： More genome stability and DNA repair genes were up-regulated in the members of the NPC clustering families than the NPC patients, and the range of up-regulated was high, with the over up-regulated 100 times genes including TEP1, MSH4, PMS2LI. Fewer genome stability and DNA repair genes were down-regulated in the members of the NPC clustering families than the NPC patients, the ubiquitin genes almost were down-regulated, the results also could be confirmed by real-time fluorescence quantitative PCR. Conclusion： There are specially expression character of genome stability and DNA repair genes in the members of NPC clustering families.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(No.2012AA10A409)the Scientific and Technological Innovation Project of Qingdao National Laboratory for Marine Science and Technology(No.2015ASKJ02)
文摘The transformer-2(tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster.In this study,sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized.Four tra-2 isoforms,designated as Estra-2a,Estra-2b,Estra-2c,and Estra-2d,were isolated.They all contained an RNA-recognition motif(RRM) and a linker region,which shared high similarity with other reported tra-2s.Sequence analysis revealed that Estra-2a,Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA.Compared with the other three isoforms,Estra-2d lacks the RS2 domain.Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg,and in the 2-4 cell and blastula stages compared with larval stages(P<0.01),suggesting their maternal origin in early embryonic developmental stages.Notably,Estra-2a was highly expressed in male somatic tissues,while Estra-2c was significantly highly expressed in the ovary.These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab.Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.
