Accurate quantification of transcripts using quantitative real-time polymerase chain reaction （qPCR） depends on the identification of reliable reference genes for normalization. This study aimed to identify and vali...Accurate quantification of transcripts using quantitative real-time polymerase chain reaction （qPCR） depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 （ACT-2）, elongation factor 1 alpha （EF-1α）, elongation factor 1 beta （EF-1β）, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, ubiquitin （UBQ）, β-tubulin （β-TUB）, and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene （Gβ） across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve.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
【目的】通过荧光定量PCR技术对土壤微生物目标基因进行绝对定量,其定量结果的准确性容易受到DNA提取得率以及腐殖酸抑制性的影响。【方法】采用内参基因加标法,利用构建的突变质粒DNA,对供试水稻土壤样品中的微生物16S r RNA目标基因...【目的】通过荧光定量PCR技术对土壤微生物目标基因进行绝对定量,其定量结果的准确性容易受到DNA提取得率以及腐殖酸抑制性的影响。【方法】采用内参基因加标法,利用构建的突变质粒DNA,对供试水稻土壤样品中的微生物16S r RNA目标基因的绝对拷贝数进行荧光定量PCR检测,用来表征该样品中细菌群落总体丰度。在定量前通过双向引物扩增方法验证突变质粒中的内参基因对供试土壤的特异性。【结果】不同水稻土壤样品的DNA提取量在样品间差异较大。通过内参基因加标法对DNA提取量进行校正,显著提高了16S r RNA基因绝对定量的精确度。不同水稻土壤样品间的变异系数为17.8,与未加标处理相比降低了66.7%。在此基础上,进一步通过内参基因加标法对土壤有机质和含水率均呈现典型空间特征差异的6处亚热带湿地土壤样品中的16S r RNA基因进行绝对定量。16S r RNA基因绝对拷贝数与土壤微生物生物量碳具有显著的线性相关性(R2=0.694,P<0.001),表明内参校正后的16S r RNA基因绝对拷贝数可以准确反映单位质量土壤中微生物的丰度。【结论】内参基因加标法可以对DNA提取得率以及腐殖酸对PCR扩增的抑制性进行校正,从而提高绝对定量的准确性。基于内参基因加标法的目标基因绝对定量PCR检测,可作为土壤微生物生物量测量,以及微生物功能基因绝对丰度定量的一种核酸检测方法。展开更多
[Objective] The aim was to select suitable gene for Chlorella identification and to identify the oil-producing microalgae.[Method] Four candidate gene sequences,the nuclear genomic rDNA of the 18S rRNA gene,internal t...[Objective] The aim was to select suitable gene for Chlorella identification and to identify the oil-producing microalgae.[Method] Four candidate gene sequences,the nuclear genomic rDNA of the 18S rRNA gene,internal transcribed spacer（ITS）,internal transcribed spacer Ⅱ（ITS Ⅱ）and the chloroplast rbcL gene,were selected for Chlorella molecular identification.Through these four candidate genes,the genetic variability and distinguish ability between intra-species and inter-species was analyzed to choose the right genes for identification of the high oil-content Chlorella.On this basis,application of these gene segments were classified and identified for five fresh-water isolated Chlorella,which oil-content is more than 30%.[Result] ITS gene was a suitable gene because of its high variation and short fragment length,meanwhile its genetic distance intra-species（0.439 6±0.135 9）was larger than inter-species（0.045 7±0.084 3）.Its sequence length varied between different species whereas highly conserved in the same species.By the application of ITS sequences,respectively,five high oil-content stains were identified as one C.vulgaris,two strains of C.sorokiniana and two strains of algae Chlorella sp.[Conclusion] This study had provided reference for the establishment of identification gene pool of Chlorella.展开更多
基金Supported by the National Natural Science Foundation of China(No.41176113)the National Basic Research Program of China(973 Program)(No.2010CB126403)+1 种基金the Changjiang Scholars Program for Innovative Research Team in Universities(No.IRT0941)the Earmarked Fund for Modern Agro-Industry Technology Research System(No.nycytx-47)
文摘Accurate quantification of transcripts using quantitative real-time polymerase chain reaction （qPCR） depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 （ACT-2）, elongation factor 1 alpha （EF-1α）, elongation factor 1 beta （EF-1β）, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, ubiquitin （UBQ）, β-tubulin （β-TUB）, and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene （Gβ） across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve.
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【目的】通过荧光定量PCR技术对土壤微生物目标基因进行绝对定量,其定量结果的准确性容易受到DNA提取得率以及腐殖酸抑制性的影响。【方法】采用内参基因加标法,利用构建的突变质粒DNA,对供试水稻土壤样品中的微生物16S r RNA目标基因的绝对拷贝数进行荧光定量PCR检测,用来表征该样品中细菌群落总体丰度。在定量前通过双向引物扩增方法验证突变质粒中的内参基因对供试土壤的特异性。【结果】不同水稻土壤样品的DNA提取量在样品间差异较大。通过内参基因加标法对DNA提取量进行校正,显著提高了16S r RNA基因绝对定量的精确度。不同水稻土壤样品间的变异系数为17.8,与未加标处理相比降低了66.7%。在此基础上,进一步通过内参基因加标法对土壤有机质和含水率均呈现典型空间特征差异的6处亚热带湿地土壤样品中的16S r RNA基因进行绝对定量。16S r RNA基因绝对拷贝数与土壤微生物生物量碳具有显著的线性相关性(R2=0.694,P<0.001),表明内参校正后的16S r RNA基因绝对拷贝数可以准确反映单位质量土壤中微生物的丰度。【结论】内参基因加标法可以对DNA提取得率以及腐殖酸对PCR扩增的抑制性进行校正,从而提高绝对定量的准确性。基于内参基因加标法的目标基因绝对定量PCR检测,可作为土壤微生物生物量测量,以及微生物功能基因绝对丰度定量的一种核酸检测方法。
基金Supported by Key Project of Knowledge Innovation Project of Chinese Academy of Sciences(KGCX2-YW-374-3)Scientific and Technological Project of Shandong Province(2008GG20007002)~~
文摘[Objective] The aim was to select suitable gene for Chlorella identification and to identify the oil-producing microalgae.[Method] Four candidate gene sequences,the nuclear genomic rDNA of the 18S rRNA gene,internal transcribed spacer（ITS）,internal transcribed spacer Ⅱ（ITS Ⅱ）and the chloroplast rbcL gene,were selected for Chlorella molecular identification.Through these four candidate genes,the genetic variability and distinguish ability between intra-species and inter-species was analyzed to choose the right genes for identification of the high oil-content Chlorella.On this basis,application of these gene segments were classified and identified for five fresh-water isolated Chlorella,which oil-content is more than 30%.[Result] ITS gene was a suitable gene because of its high variation and short fragment length,meanwhile its genetic distance intra-species（0.439 6±0.135 9）was larger than inter-species（0.045 7±0.084 3）.Its sequence length varied between different species whereas highly conserved in the same species.By the application of ITS sequences,respectively,five high oil-content stains were identified as one C.vulgaris,two strains of C.sorokiniana and two strains of algae Chlorella sp.[Conclusion] This study had provided reference for the establishment of identification gene pool of Chlorella.
