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Comparison of next generation sequencing-based and methylated DNA immunoprecipitation-based approaches for fetal aneuploidy non-invasive prenatal testing

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World Journal of Medical Genetics 2015年第2期5卷 23-27页

作者： Georgia Christopoulou Elisavet A Papageorgiou Philippos C Patsalis Voula Velissariou Alfa Lab Cytogenetics and Molecular Biology Center Hygeia Group NIPD Genetics Ltd. the Cyprus Institute of Neurology and Genetics Ministry of Health Government Office

Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorionic villus and amniotic fluid sampling, which result in a significant risk for pregnancy loss. The discovery of cell free fetal DNA circulating in the maternal blood has great potential for the development of non-invasive prenatal testing(NIPT) methodologies. Such strategies have been successfully applied for the determination of the fetal rhesus status and inherited monogenic disease but the field of fetal aneuploidy investigation seems to be more challenging. The main reason for this is that the maternal cell free DNA in the mother's plasma is far more abundant, and because it is identical to half of the corresponding fetal DNA. Approaches developed are mainly based on next generation sequencing(NGS) technologies and epigenetic genetic modifications, such as fetal-maternal DNA differential methylation. At present, genetic services for non-invasive fetal aneuploidy detection are offered using NGS-based approaches but, for reasons that are presented herein, they still serve as screening tests which are not readily accessed by the majority of couples. Here we discuss the limitations of both strategies for NIPT and the future potential of the methods developed.

关键词： Next generation sequencing Differential methylation Epigenetics Fetal aneuploidy Methylation dependent immunoprecipitation Non-invasive prenatal testing

The Effect of Elution Volume for immunoprecipitation on m6A-Seq Analysis

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Proceedings of Anticancer Research 2021年第4期5卷 1-5页

作者： Yuhui Xu Lize Shen Guolin Li College of Life Sciences Hunan Normal UniversityChangsha 410081Hunan ProvinceChina R&D Department LC-Bio Technology Co.LtdHangzhou 310018Zhejiang ProvinceChina

Objective:To develop a cost-effective method to reduce the time consumption of elution in *** Two volumes(125μL for Group C and 100μL for Group T)of elution buffer were used to explore whether smaller volume could save testing ***:Time consumption of elution in Group T was significantly shorter than that in Group C,while the efficiency of eluted m6A-containing fragments and the performance of m^(6)A-Seq as indicated by m6A peak distributions showed no difference between the two ***:A smaller volume of elution buffer was an economical way to reduce time consumption in immunoprecipitation.

关键词： m^(6)A-Seq immunoprecipitation Elution buffer

Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain

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Advances in Bioscience and Biotechnology 2012年第5期3卷 626-629页

作者： Toshitsugu Fujita Hodaka Fujii Combined Program on Microbiology and Immunology Research Institute for Microbial Diseases Osaka University Suita Osaka Japan

Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.

关键词： Insertional Chromatin immunoprecipitation iChIP LexA FLAG Tag

Verification of the Interaction between SET and eEF1A1 in Human Liver Cells by Co-immunoprecipitation

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Agricultural Science & Technology 2010年第8期11卷 87-90页

作者：杨亮杨细飞张毅刘建军李杰华南理工大学生物科学与工程学院广东广州510006 深圳市疾病预防控制中心毒理研究室广东深圳518020

[Objective] The aim was to investigate the possible interaction between SET and eEF1A1 in human liver cells. [Method] Firstly the total proteins of human L-02 liver cells were extracted under non-denaturing conditions; then,mouse anti-human SET and rabbit anti-human eEF1A1 antibodies were used to perform the co-immunoprecipitation respectively; subsequently,the immunoprecipitations was correspondingly detected with rabbit anti-human eEF1A1 and mouse anti-human SET antibodies by Western Blot. [Result] EEF1A1 was detected in protein complex from the immunoprecipitations by using anti-SET antibody,and SET also was detected in immunoprecipitations by using anti-eEF1A1 antibody. [Conclusion] The interaction between SET and eEF1A1 in human liver cells was confirmed.

关键词： SET eEF1A1 immunoprecipitation Interaction

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Easy-to-use Method to Display Arabidopsis Rubisco Interacting Proteins

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Chemical Research in Chinese Universities 2010年第3期26卷 427-430页

作者： SUN Li-wen LI Jian-hua LI Shan-yu XI Jing-hui Key Lab for Molecular Enzymology and Engineering Ministry of Education Jilin University Changchun 130021 P. R. China Department of Engineering Chemistry Mudanjiang University Mudanjiang 157011 P. R. China Department of Pediatrics The First Hospital of Jilin University Changehun 130021 P. R. China College of Plant Science Jilin University Changehun 130062 P. R. China

Affinity-mediated protein separation is an integral part of proteomics, the most outstanding of which is immunoproteomics. However, in the immunoprecipitate system overwhelming Ab and Ag conceal Ag interacting proteins as the research targets, which is the rate-limiting step in the progress of comparative proteomic analyses. We presented a convenient and accurate method to tackle this problem. 1 mol/L NaCl elution buffer was applied to the complex Rubisco immunoprecipitate of Arabidopsis, the weakest force involved in the system was selectively broken up, resulting in the enrichment of Rubisco interacting proteins accessible for further comparative protein gel profile. The easy-to-use method sheds light on a narrow-down strategy supplement for comparative immunoproteomics.

关键词： Comparative proteomics Arabidopsis Rubisco immunoprecipitation

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Characterization of endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2 cells

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BIOCELL 2022年第2期46卷 547-557页

作者： LIXU JIANG LIN NING CHUNCHAO PU ZIXIN WANG BIFANG HE JIAN HUANG School of Life Science and Technology University of Electronic Science and Technology of ChinaChengdu611731China School of Healthcare Technology Chengdu Neusoft UniversityChengdu611844China School of Medicine Guizhou UniversityGuiyang550025China

As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable *** gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very *** now,the endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2(*** sp2)have not been *** expressed the conserved PIWI domain of NgAgo and used it to induce anti-PIWI *** also cultured the *** sp2 strain and performed immunoprecipitation,chromatin immunoprecipitation(ChIP),and RNA immunoprecipitation(RIP)*** nucleic acids that endogenously bound NgAgo in *** sp2 cells were sequenced and *** results showed that NgAgo endogenously bound RNA rather than ***-associated RNAs were mainly transcripts of genes that encoded tRNA,transcriptional regulators,RNA polymerases,and RNA-binding *** mainly binds to the transcripts inside genes or in their upstream ***,the top enriched motif of peaks was the same as that of miR-1289,suggesting that NgAgo may regulate gene expression *** enrichment analysis showed that the peak-associated genes were enriched in transmembrane transport *** results revealed that NgAgo binds RNA and may function in post-transcriptional regulation in vivo.

关键词： NgAgo PIWI immunoprecipitation ChIP-seq RIP-seq Post-transcriptional regulation

Identification of new centrosome proteins by autoimmune patient sera

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Science China(Life Sciences) 2007年第2期50卷 194-202页

作者： XIA Liang1, LI Yan1, YANG Dong1, WANG LiMin1, HE Fang1, ZHOU ChunYuan1, LI YongZhe2, ZENG ChangQing1 & He DaCheng1 1 Key Laboratory for Cell Proliferation and Regulation Biology of Ministry of Education, Beijing Normal University, Beijing 100875, China 2 Peking Union Medical Hospital, Peking Union Medical College, Beijing 100730, China 3 Beijing Genomic Institute, Beijing 101300, China 1. Key Laboratory for Cell Proliferation and Regulation Biology of Ministry of Education Beijing Normal University Beijing 100875 China 2. Peking Union Medical Hospital Peking Union Medical College Beijing 100730 China

Compared to other subcellular organelles, centrosome proteome can hardly be studied, due to the dif- ficulties in separation and purification of centrosome. Auto-antisera from 6 autoimmune patients, which recognized centrosome specifically in immunofluorescence, were used to identify the corresponding centrosomal proteins. The sera were first tested by Western blot on whole cell lysate, and all bound antibodies were then eluted from each single band in Western blot membrane to assure which antibody was responsible for the centrosome specific immunofluorescence staining. The corresponding pro- teins were obtained by immunoprecipitation and identified by mass spectrometry. Six centrosomal proteins, including 2 known centrosomal proteins and 4 proteins with unknown localization or report- edly non-centrosomal localization, were identified. These proteins apparently involve in cell cycle regulation, signal transduction pathways, molecular chaperons, and metabolism enzymes, which may reflect the expected functional diversity of centrosome.

关键词： centrosome autoimmune patient sera elution of antibody immunoflourescence immunoprecipitation mass spectrometry

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Identification of target genes of transcription factor CEBPB in acute promyelocytic leukemia cells induced by all-trans retinoic acid

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Asian Pacific Journal of Tropical Medicine 2013年第6期6卷 473-480页

作者： Lei Yu Yang-De Zhang Jun Zhou De-Ming Yao Xiang Li Department of Hepatic Surgery National Hepatobiliary and Enteric Surgery Research CenterMinistry of HealthCentral South UniversityChina

Objective:To indentify target genes of transcription factor CCA AT enhancer-binding protein P(CEBPB) in acute proinyelocytie leukemia cells induced by all-tram retinoie ***: A new strategy for high—throughput identification of direct target genes was established by combining chromatin immunoprecipitation(ChIP) with in vitro ***,106 potential CKBPB binding fragments from the genome of the all-trans retinoie acid(ATRA)-treated NB4 cells were ***:Of them,82 were mapped in proximity to known or previously predicted genes;7 were randomly picked up for further confirmation by ChlP-PCR and 3 genes (CALM,1TPR2 and 0RM2) were found to be specificaUy up-regulated in the ATRA-treated NB4 cells,indicating that they might lie the down-stream target genes of ***:Our results provided new insight into the mechanisms of ATRA-induced granulocytic differentiation.

关键词： Chromatin immunoprecipitation All-trans retinoic acid CEBP Granulopoiesis Neutrophils

A Brassinosteroid-Signaling Kinase Interacts withMultiple Receptor-Like Kinases in Arabidopsis

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Molecular Plant 2014年第2期7卷 441-444页

作者： Peng Xu[a,b] Shou-Ling Xu[b,c] Zi-Jing Li[a] Wenqiang Tang[a] Alma L. Burlingame[c] Zhi-Yong Wang[b] [a]Institute of Molecular and Cell Biology Hebei Key Laboratory of Molecular and Cellular Biology College of Life Sciences Hebei Normal University Shijiazhuang Hebei 050024 China [b]Department of Plant Biology Carnegie Institution for Science Stanford CA 94305 USA [c]Department of Pharmaceutical Chemistry University of California San Francisco CA 94143 USA

Dear Editor, Higher plants have evolved hundreds of genes encodingreceptor-like kinases （RLKs）, which function as cell surfacereceptors perceiving developmental and environmental sig-nals （Shiu et al., 2004）. Many RLKs have been shown to playspecific roles in hormone responses, developmental regula-tion, defense against pathogen infection, and adaptationto abiotic stresses （Chae et al., 2009; Antolin-Llovera et al.,2012）. The mechanisms that ensure specific signal transduc-tion from each RLK to target cellular responses remain poorlyunderstood. Recent studies revealed that many RLKs trans-duce signals by phosphorylating receptor-like cytoplasmickinases （RLCKs）, which lack the transmembrane domainsbut are anchored at the plasma membrane through lipidmodification （Tang et al., 2008; Zhang et al., 2010; Shi et al.,2013）. There are over 400 RLKs and only about 150 RLCKs inArabidopsis （Shiu et al., 2004）. One outstanding question iswhether each RLCK mediates signaling downstream of a spe-cific RLK, participates in multiple RLK pathways, or mediatescrosstalk between RLK pathways.

关键词： BRASSINOSTEROIDS RESEARCH PLANT hormones PLANT cellular signal transduction CELL receptors ARABIDOPSIS immunoprecipitation NICOTIANA benthamiana