Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP)
| Autor: | Renzo J. M. Riemens, Gunter Kenis, Jennifer Nolz, Sonia C. Susano Chaves, Diane Duroux, Ehsan Pishva, Diego Mastroeni, Kristel Van Steen, Thomas Haaf, Daniël L. A. van den Hove |
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| Přispěvatelé: | Basic Neuroscience 1, RS: MHeNs - R3 - Neuroscience, Psychiatrie & Neuropsychologie, MUMC+: MA Niet Med Staf Psychiatrie (9) |
| Rok vydání: | 2022 |
| Předmět: |
Biochemistry & Molecular Biology
OOCYTES Chemistry Multidisciplinary laser-capture microdissection Catalysis Inorganic Chemistry limiting dilution bisulfite pyrosequencing Humans HETEROGENEITY MESSENGER-RNA EXPRESSION Physical and Theoretical Chemistry Molecular Biology Spectroscopy Science & Technology DNA methylation epigenetics Lasers Organic Chemistry Brain High-Throughput Nucleotide Sequencing Neurodegenerative Diseases General Medicine single-cell DNA Methylation Computer Science Applications DNA/methods Chemistry Sequence Analysis DNA/methods Physical Sciences PATTERNS Apoptosis Regulatory Proteins Sequence Analysis Life Sciences & Biomedicine Molecular Chaperones |
| Zdroj: | International journal of molecular sciences, 23(24):15571. Multidisciplinary Digital Publishing Institute (MDPI) International Journal of Molecular Sciences; Volume 23; Issue 24; Pages: 15571 International Journal of Molecular Sciences |
| ISSN: | 1661-6596 |
| DOI: | 10.5281/zenodo.7468395 |
| Popis: | 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. https://doi.org/10.3390/ijms232415571 This research was funded by by the Joint Programme—Neurodegenerative Disease Re- search (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 inno- vation 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). |
| Databáze: | OpenAIRE |
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