Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP)

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*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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.

Original languageEnglish
Article number15571
Number of pages18
JournalInternational journal of molecular sciences
Volume23
Issue number24
DOIs
Publication statusPublished - 8 Dec 2022

Keywords

  • Apoptosis Regulatory Proteins
  • Brain
  • DNA Methylation
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Lasers
  • Molecular Chaperones
  • Neurodegenerative Diseases
  • Sequence Analysis, DNA/methods

Fingerprint

Dive into the research topics of 'Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP)'. Together they form a unique fingerprint.

Cite this