Article (Scientific journals)
Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP).
Riemens, Renzo J M; Kenis, Gunter; Nolz, Jennifer et al.
2022In International Journal of Molecular Sciences, 23 (24), p. 15571
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Keywords :
DNA methylation; epigenetics; laser-capture microdissection; limiting dilution bisulfite pyrosequencing; single-cell; hydrogen sulfite; DNAJB13 protein, human; Molecular Chaperones; Apoptosis Regulatory Proteins; Humans; Sequence Analysis, DNA/methods; DNA Methylation; Brain; High-Throughput Nucleotide Sequencing; Lasers; Neurodegenerative Diseases; Sequence Analysis, DNA; Catalysis; Molecular Biology; Spectroscopy; Computer Science Applications; Physical and Theoretical Chemistry; Organic Chemistry; Inorganic Chemistry; General Medicine
Abstract :
[en] A reoccurring issue in neuroepigenomic studies, especially in the context of neurodegenerative disease, is the use of (heterogeneous) bulk tissue, which generates noise during epigenetic profiling. A workable solution to this issue is to quantify epigenetic patterns in individually isolated neuronal cells using laser capture microdissection (LCM). For this purpose, we established a novel approach for targeted DNA methylation profiling of individual genes that relies on a combination of LCM and limiting dilution bisulfite pyrosequencing (LDBSP). Using this approach, we determined cytosine-phosphate-guanine (CpG) methylation rates of single alleles derived from 50 neurons that were isolated from unfixed post-mortem brain tissue. In the present manuscript, we describe the general workflow and, as a showcase, demonstrate how targeted methylation analysis of various genes, in this case, RHBDF2, OXT, TNXB, DNAJB13, PGLYRP1, C3, and LMX1B, can be performed simultaneously. By doing so, we describe an adapted data analysis pipeline for LDBSP, allowing one to include and correct CpG methylation rates derived from multi-allele reactions. In addition, we show that the efficiency of LDBSP on DNA derived from LCM neurons is similar to the efficiency obtained in previously published studies using this technique on other cell types. Overall, the method described here provides the user with a more accurate estimation of the DNA methylation status of each target gene in the analyzed cell pools, thereby adding further validity to this approach.
Disciplines :
Life sciences: Multidisciplinary, general & others
Author, co-author :
Riemens, Renzo J M ;  Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, 6229 ER Maastricht, The Netherlands ; Institute of Human Genetics, Julius Maximilians University, 97074 Wuerzburg, Germany
Kenis, Gunter;  Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, 6229 ER Maastricht, The Netherlands
Nolz, Jennifer;  Biodesign Institute, Neurodegenerative Disease Research Center, Arizona State University, Tempe, AZ 85281, USA
Susano Chaves, Sonia C ;  Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, 6229 ER Maastricht, The Netherlands
Duroux, Diane  ;  Université de Liège - ULiège > GIGA > GIGA Medical Genomics - Biostatistics, biomedicine and bioinformatics
Pishva, Ehsan;  Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, 6229 ER Maastricht, The Netherlands
Mastroeni, Diego;  Biodesign Institute, Neurodegenerative Disease Research Center, Arizona State University, Tempe, AZ 85281, USA
Van Steen, Kristel  ;  Université de Liège - ULiège > Département d'électricité, électronique et informatique (Institut Montefiore) > Bioinformatique ; BIO3 Laboratory for Systems Medicine, Department of Human Genetics, KU Leuven, 3000 Leuven, Belgium
Haaf, Thomas ;  Institute of Human Genetics, Julius Maximilians University, 97074 Wuerzburg, Germany
van den Hove, Daniël L A ;  Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, 6229 ER Maastricht, The Netherlands ; Laboratory of Translational Neuroscience, Department of Psychiatry, Psychosomatics and Psychotherapy, University of Wuerzburg, 97080 Wuerzburg, Germany
Language :
English
Title :
Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP).
Publication date :
08 December 2022
Journal title :
International Journal of Molecular Sciences
ISSN :
1661-6596
eISSN :
1422-0067
Publisher :
MDPI, Switzerland
Volume :
23
Issue :
24
Pages :
15571
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
ZonMw - Netherlands Organisation for Health Research and Development [NL]
MRC - Medical Research Council [GB]
BMBF - Federal Ministry of Education and Research [DE]
FNR - Luxembourg National Research Fund [LU]
Funding text :
This research was funded by by the Joint Programme—Neurodegenerative Disease Research (JPND) for the EPIAD consortium (http://www.neurodegenerationresearch.eu/wp-content/uploads/2015/10/Factsheet_EPI-AD.pdf) (DvdH). The project is supported through the following funding organizations under the aegis of JPND; The Netherlands, The Netherlands Organisation for Health Research and Development (ZonMw); United Kingdom, Medical Research Council; Germany, German Federal ministry of Education and Research (BMBF); Luxembourg, National Research Fund (FNR). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 643417. Additional funds have been provided by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreements No. 813533 and No. 860895, and under Grant Agreement No. 643417 (KVS).
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