| Autor: |
Riemens RJM; 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 G; Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, 6229 ER Maastricht, The Netherlands., Nolz J; Biodesign Institute, Neurodegenerative Disease Research Center, Arizona State University, Tempe, AZ 85281, USA., Susano Chaves SC; Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, 6229 ER Maastricht, The Netherlands., Duroux D; BIO3 Laboratory for Systems Genetics, GIGA-R Medical Genomics, University of Liège, 4000 Liège, Belgium., Pishva E; Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, 6229 ER Maastricht, The Netherlands., Mastroeni D; Biodesign Institute, Neurodegenerative Disease Research Center, Arizona State University, Tempe, AZ 85281, USA., Van Steen K; BIO3 Laboratory for Systems Genetics, GIGA-R Medical Genomics, University of Liège, 4000 Liège, Belgium.; BIO3 Laboratory for Systems Medicine, Department of Human Genetics, KU Leuven, 3000 Leuven, Belgium., Haaf T; Institute of Human Genetics, Julius Maximilians University, 97074 Wuerzburg, Germany., van den Hove DLA; 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. |
| Abstrakt: |
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. |
