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
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
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.
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.
