AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl trans...AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.展开更多
The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we esta...The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.展开更多
The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization....The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.展开更多
The sheep genome harbours approximately 20 copies of endogenous beta-retroviruses （enJSRVs）, and circumstantial evidence suggests that enJSRVs might play a role in mammalian reproduction, particularly placental morp...The sheep genome harbours approximately 20 copies of endogenous beta-retroviruses （enJSRVs）, and circumstantial evidence suggests that enJSRVs might play a role in mammalian reproduction, particularly placental morphogenesis. This study was aimed to assess the expression of mRNAs of an enJSRV and its receptor, HYAL2, in the uterus and conceptuses of Mongolian ewes throughout gestation, using real-time reverse transcription polymerase chain reaction and in situ hybridization analysis. The results showed that enJSRV and HYAL2 mRNAs were found to be expressed throughout gestation in the endometrium, chorion, placenta, and conceptus. The enJSRV mRNA was most abundant in the placenta on day 90 of pregnancy, in the endometrium on day 30 and 50, and in the chorion on day 70 and 110. However, HYAL2 mRNA was most abundant in the endometrium on day 30. These differences were all significantly different from each other （P〈0.01）. In situ hybridization showed that enJSRV and HYAL2 mRNAs were specifically expressed in endometrial luminal epithelium and glandular epithelium, trophoblastic giant binucleated cells （BNCs）, endometrial caruncles, placental cotyledons, stroma, trophectoderm, as well as multinucleated syncytia of the placenta and blood vessel endothelial cells. Collectively, little is known about the molecular mechanisms by which trophoblastic differentiation and multinucleated syncytia formation are regulated by enJSRVs. However, the temporal and spatial distributions of enJSRV expression in the uterus and conceptus indicate that differentiation of BNCs and the formation of a multinucleated syncytiotrophoblast involve enJSRV and possibly its cellular receptor, HYAL2. Therefore, enJSRV and HYAL2 appear to play important roles in the female reproductive physiology in this breed of sheep.展开更多
Drought is one of the major abiotic stresses which adversely affect crop plants limiting growth and yield potential.Structural and functional characterization of drought stress-induced genes has contributed to a bette...Drought is one of the major abiotic stresses which adversely affect crop plants limiting growth and yield potential.Structural and functional characterization of drought stress-induced genes has contributed to a better understanding of how plants respond and adapt to the drought stress.In the present study,differential display technique was employed to study the gene expression of rice plants at the reproductive stage that were subjected to drought stress by withholding water,Pseudomonas fluorescens strain(Pf1) treated plants subjected for drought stress by withholding water and control(well-watered).Differentially expressed c DNAs of six genes(COX1,PKDP,b ZIP1,AP2-EREBP,Hsp20 and COC1) were identified,cloned and sequenced.Real-time q PCR analysis showed that all the six genes were upregulated in drought-stressed plants treated with Pf1.This revealed that the remarkable influence of Pf1 colonization leads to drought tolerance at the reproductive stage.These results showed that high levels of gene expression in plants lacking adequate water can be remarkably influenced by Pf1 colonization,which might be a key element for induced systemic tolerance by microbes.展开更多
There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research.In ...There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research.In this study,we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes:Actb,Ankrd27,CypA,Gapdh,Hprt1,Mrpl10,Pgk1,Rictor,Rn18s,Tbp,Ubc,Ubxn11,and Ywhaz.Our results show that the expression of Ankrd27,Ubc,and Tbp were stable after spinal cord injury,while Actb was the most unstable internal control gene.Ankrd27,Ubc,Tbp,and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression.Target gene expression levels and changes over time were similar when Ankrd27,Ubc,and Tbp were used as internal controls but different when Actb was used as an internal control.We recommend that Ankrd27,Ubc,and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research.This study was approved by the Administration Committee of Experimental Animals,Jiangsu Province,China(approval No.20180304-008)on March 4,2018.展开更多
Objective: The aim of this study was to determine the etiology and prevalence of pediatric CAP in Beijing using a real-time polymerase chain reaction (PCR) technique. Methods: Between February 15, 2011 and January 18...Objective: The aim of this study was to determine the etiology and prevalence of pediatric CAP in Beijing using a real-time polymerase chain reaction (PCR) technique. Methods: Between February 15, 2011 and January 18, 2012, 371 pediatric patients with CAP were enrolled at Beijing Children's Hospital. Sixteen respiratory viruses and two bacteria were detected from tracheal aspirate specimens using commercially available multiplex real-time reverse transcription PCR (RT-PCR) kits. Results: A single viral pathogen was detected in 35.3%of enrolled patients, multiple viruses in 11.6%, and virus/bacteria co-infection in 17.8%. In contrast, only 6.5%of patients had a single bacterial pathogen and 2.2%were infected with multiple bacteria. The etiological agent was unknown for 26.7% of patients. The most common viruses were respiratory syncytial virus (RSV) (43.9%), rhinovirus (14.8%), parainfluenza virus (9.4%), and adenovirus (8.6%). In patients under three years of age, RSV (44.6%), rhinovirus (12.8%), and Streptococcus pneumoniae (9.9%) were the most frequent pathogens. In children aged 3e7 years, S. pneumoniae (38.9%), RSV (30.6%), Haemophilus influenzae (19.4%), and adenovirus (19.4%) were most prevalent. Finally in children over seven years, RSV (47.3%), S. pneumoniae (41.9%), and rhinovirus (21.5%) infections were most frequent. Conclusions: Viral pathogens, specifically RSV, were responsible for the majority of CAP in pediatric patients. However, both S. pneumoniae and H. influenzae contributed as major causes of disease. Commercially available multiplexing real-time PCR allowed for rapid detection of the etiological agent. Copyright ? 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).展开更多
Aim:Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)is still the“gold standard”for quantitative analysis of mRNA and the study of differentially expressed genes.Methods:The authors describe a RT...Aim:Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)is still the“gold standard”for quantitative analysis of mRNA and the study of differentially expressed genes.Methods:The authors describe a RT-qPCR array that exploits SYBR Green dye-based detection to perform reliable gene expression analysis on 41 genes involved in several pathways linked to DNA damage response,cell cycle progression,cellular senescence,and programmed cell death.To validate the RT-qPCR array,the authors investigated changes of the gene expression profile of HeLa cells treated with two well-characterized antiproliferative molecules such as cisplatin(CDDP)and sodium butyrate(NaBu).Results:The results showed a gene expression profile compatible with both biological and gene expression data already reported in literature.Conclusion:Importantly,the assay allowed the monitoring of additional and not reported gene regulations,indicating that this custom-made RT-qPCR array is a cheap,robust,and rapid tool for the study of drug-induced effects in human biological models.展开更多
Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse t...Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR （RT-PCR）, and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-l, jmjdla, jmjd2c, and cyclin DI. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expression significantly decreased, whereas expression of neuronal markers, such as microtubule associated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.展开更多
Objective This study aimed to investigate the expression and significance of PIN2/TERF1 interacting, telomerase inhibitor 1(Pin X1) and human telomerase reverse transcriptase(h TERT) in basal cell carcinoma(BCC). Meth...Objective This study aimed to investigate the expression and significance of PIN2/TERF1 interacting, telomerase inhibitor 1(Pin X1) and human telomerase reverse transcriptase(h TERT) in basal cell carcinoma(BCC). Methods Real-time polymerase chain reaction and immunohistochemistry were performed to quantify the m RNA expressions and integrated optical density(IOD), respectively, of Pin X1 and h TERT in BCC specimens(n = 30), as well as in normal skin specimens(n = 15). Results The m RNA expression level and IOD of Pin X1 in the BCC samples were both significantly lower than those in the control specimens(P < 0.05). Conversely, the m RNA expression level and IOD of h TERT in BCC were both significantly higher than that in the control samples(P < 0.05). The correlation between the expression levels of Pin X1 and h TERT showed no statistical significance(P > 0.05). Conclusion Downregulation of Pin X1 and upregulation of h TERT expression may be associated with the activation and maintenance of telomerases in the induction of BCC.展开更多
The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(c...The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(cp)gene of Cymbidium mosaic virus(CyMV).The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity.The standard curve was drawn based on the linear relationship between the logarithm(base 10)of the quantity of target sequence and cycle threshold[C(T)].While the concentration of plasmid DNA falling within the range of 2.6×10^(7)to 2.6×10^(2)copies per tube established a regression equation,y=-0.3583x+10.32,and related coefficient:r^(2)=0.995.The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube.The naturally infected samples of Phalaenopsis sp.and the artificially inoculated samples of Arachnis sp.with trace CyMV were quantitatively detected using this method.CyMVin the positive samples of Phalaenopsis sp.and Arachnis sp.was confirmed by DNA sequencing and cp gene homeology blast.The results showed that CyMV extracted from the leaves of orchid in Hangzhou,Zhejiang Province,China,could be derived from Kunming city(KM),Yunnan Province,China.This method characterized by high sensitivity,specificity,and precision is suitable for early diagnosis and quantitative detection of CyMV.展开更多
基金Supported by grant from Fundamental Research Grant Scheme by Ministry of Higher Education(MoHE)600-IRMI/FRGS 5/3(101/2019).
文摘AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.
基金supported by the National Natural Science Foundation of China (Nos. 31470271 and 81730110)Guangzhou Science and Technology Program key projects (No. 201803040006)
文摘The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.
基金Supported by the Key Program of Science and Technology Innovation in Ningbo (No.2019B10009)the National Natural Science Foundation of China (No.41476111)。
文摘The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.
基金funded by the National Natural Science Foundation of China (30960271 and 31160493)the doctor fund project of Ministry of Education of China(20111515110008)
文摘The sheep genome harbours approximately 20 copies of endogenous beta-retroviruses （enJSRVs）, and circumstantial evidence suggests that enJSRVs might play a role in mammalian reproduction, particularly placental morphogenesis. This study was aimed to assess the expression of mRNAs of an enJSRV and its receptor, HYAL2, in the uterus and conceptuses of Mongolian ewes throughout gestation, using real-time reverse transcription polymerase chain reaction and in situ hybridization analysis. The results showed that enJSRV and HYAL2 mRNAs were found to be expressed throughout gestation in the endometrium, chorion, placenta, and conceptus. The enJSRV mRNA was most abundant in the placenta on day 90 of pregnancy, in the endometrium on day 30 and 50, and in the chorion on day 70 and 110. However, HYAL2 mRNA was most abundant in the endometrium on day 30. These differences were all significantly different from each other （P〈0.01）. In situ hybridization showed that enJSRV and HYAL2 mRNAs were specifically expressed in endometrial luminal epithelium and glandular epithelium, trophoblastic giant binucleated cells （BNCs）, endometrial caruncles, placental cotyledons, stroma, trophectoderm, as well as multinucleated syncytia of the placenta and blood vessel endothelial cells. Collectively, little is known about the molecular mechanisms by which trophoblastic differentiation and multinucleated syncytia formation are regulated by enJSRVs. However, the temporal and spatial distributions of enJSRV expression in the uterus and conceptus indicate that differentiation of BNCs and the formation of a multinucleated syncytiotrophoblast involve enJSRV and possibly its cellular receptor, HYAL2. Therefore, enJSRV and HYAL2 appear to play important roles in the female reproductive physiology in this breed of sheep.
基金the Jawaharlal Nehru University(JNU) research fellowship sponsored by the Department of Biotechnology(DBT),Government of India
文摘Drought is one of the major abiotic stresses which adversely affect crop plants limiting growth and yield potential.Structural and functional characterization of drought stress-induced genes has contributed to a better understanding of how plants respond and adapt to the drought stress.In the present study,differential display technique was employed to study the gene expression of rice plants at the reproductive stage that were subjected to drought stress by withholding water,Pseudomonas fluorescens strain(Pf1) treated plants subjected for drought stress by withholding water and control(well-watered).Differentially expressed c DNAs of six genes(COX1,PKDP,b ZIP1,AP2-EREBP,Hsp20 and COC1) were identified,cloned and sequenced.Real-time q PCR analysis showed that all the six genes were upregulated in drought-stressed plants treated with Pf1.This revealed that the remarkable influence of Pf1 colonization leads to drought tolerance at the reproductive stage.These results showed that high levels of gene expression in plants lacking adequate water can be remarkably influenced by Pf1 colonization,which might be a key element for induced systemic tolerance by microbes.
基金the National Natural Science Foundation of China,No.81901257(to YXW)the Natural Science Foundation of Jiangsu Province of China,No.BK20180951(to YXW)+1 种基金Postgraduate Research and Practice Innovation Program of Jiangsu Province of China,No.KYCX20_2818(to WL)and Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD,to Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education).
文摘There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research.In this study,we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes:Actb,Ankrd27,CypA,Gapdh,Hprt1,Mrpl10,Pgk1,Rictor,Rn18s,Tbp,Ubc,Ubxn11,and Ywhaz.Our results show that the expression of Ankrd27,Ubc,and Tbp were stable after spinal cord injury,while Actb was the most unstable internal control gene.Ankrd27,Ubc,Tbp,and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression.Target gene expression levels and changes over time were similar when Ankrd27,Ubc,and Tbp were used as internal controls but different when Actb was used as an internal control.We recommend that Ankrd27,Ubc,and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research.This study was approved by the Administration Committee of Experimental Animals,Jiangsu Province,China(approval No.20180304-008)on March 4,2018.
文摘Objective: The aim of this study was to determine the etiology and prevalence of pediatric CAP in Beijing using a real-time polymerase chain reaction (PCR) technique. Methods: Between February 15, 2011 and January 18, 2012, 371 pediatric patients with CAP were enrolled at Beijing Children's Hospital. Sixteen respiratory viruses and two bacteria were detected from tracheal aspirate specimens using commercially available multiplex real-time reverse transcription PCR (RT-PCR) kits. Results: A single viral pathogen was detected in 35.3%of enrolled patients, multiple viruses in 11.6%, and virus/bacteria co-infection in 17.8%. In contrast, only 6.5%of patients had a single bacterial pathogen and 2.2%were infected with multiple bacteria. The etiological agent was unknown for 26.7% of patients. The most common viruses were respiratory syncytial virus (RSV) (43.9%), rhinovirus (14.8%), parainfluenza virus (9.4%), and adenovirus (8.6%). In patients under three years of age, RSV (44.6%), rhinovirus (12.8%), and Streptococcus pneumoniae (9.9%) were the most frequent pathogens. In children aged 3e7 years, S. pneumoniae (38.9%), RSV (30.6%), Haemophilus influenzae (19.4%), and adenovirus (19.4%) were most prevalent. Finally in children over seven years, RSV (47.3%), S. pneumoniae (41.9%), and rhinovirus (21.5%) infections were most frequent. Conclusions: Viral pathogens, specifically RSV, were responsible for the majority of CAP in pediatric patients. However, both S. pneumoniae and H. influenzae contributed as major causes of disease. Commercially available multiplexing real-time PCR allowed for rapid detection of the etiological agent. Copyright ? 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
基金supported by Associazione a Sostegno degli Studi Oncologici(ASSO),Fano(PU),Italy.
文摘Aim:Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)is still the“gold standard”for quantitative analysis of mRNA and the study of differentially expressed genes.Methods:The authors describe a RT-qPCR array that exploits SYBR Green dye-based detection to perform reliable gene expression analysis on 41 genes involved in several pathways linked to DNA damage response,cell cycle progression,cellular senescence,and programmed cell death.To validate the RT-qPCR array,the authors investigated changes of the gene expression profile of HeLa cells treated with two well-characterized antiproliferative molecules such as cisplatin(CDDP)and sodium butyrate(NaBu).Results:The results showed a gene expression profile compatible with both biological and gene expression data already reported in literature.Conclusion:Importantly,the assay allowed the monitoring of additional and not reported gene regulations,indicating that this custom-made RT-qPCR array is a cheap,robust,and rapid tool for the study of drug-induced effects in human biological models.
基金the research grant No. 1.1266 from International Centre for Science, High Technology and Environmental Sciences
文摘Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR （RT-PCR）, and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-l, jmjdla, jmjd2c, and cyclin DI. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expression significantly decreased, whereas expression of neuronal markers, such as microtubule associated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.
文摘Objective This study aimed to investigate the expression and significance of PIN2/TERF1 interacting, telomerase inhibitor 1(Pin X1) and human telomerase reverse transcriptase(h TERT) in basal cell carcinoma(BCC). Methods Real-time polymerase chain reaction and immunohistochemistry were performed to quantify the m RNA expressions and integrated optical density(IOD), respectively, of Pin X1 and h TERT in BCC specimens(n = 30), as well as in normal skin specimens(n = 15). Results The m RNA expression level and IOD of Pin X1 in the BCC samples were both significantly lower than those in the control specimens(P < 0.05). Conversely, the m RNA expression level and IOD of h TERT in BCC were both significantly higher than that in the control samples(P < 0.05). The correlation between the expression levels of Pin X1 and h TERT showed no statistical significance(P > 0.05). Conclusion Downregulation of Pin X1 and upregulation of h TERT expression may be associated with the activation and maintenance of telomerases in the induction of BCC.
文摘The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(cp)gene of Cymbidium mosaic virus(CyMV).The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity.The standard curve was drawn based on the linear relationship between the logarithm(base 10)of the quantity of target sequence and cycle threshold[C(T)].While the concentration of plasmid DNA falling within the range of 2.6×10^(7)to 2.6×10^(2)copies per tube established a regression equation,y=-0.3583x+10.32,and related coefficient:r^(2)=0.995.The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube.The naturally infected samples of Phalaenopsis sp.and the artificially inoculated samples of Arachnis sp.with trace CyMV were quantitatively detected using this method.CyMVin the positive samples of Phalaenopsis sp.and Arachnis sp.was confirmed by DNA sequencing and cp gene homeology blast.The results showed that CyMV extracted from the leaves of orchid in Hangzhou,Zhejiang Province,China,could be derived from Kunming city(KM),Yunnan Province,China.This method characterized by high sensitivity,specificity,and precision is suitable for early diagnosis and quantitative detection of CyMV.
