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; Susano Chaves, Sonia C.; Duroux, Diane; Pishva, Ehsan; Mastroeni, Diego; Van Steen, Kristel; Haaf, Thomas; van den Hove, Daniël L. A.

doi:10.3390/ijms232415571

Open AccessArticle

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

by

Renzo J. M. Riemens

^1,2

,

Gunter Kenis

¹,

Jennifer Nolz

³,

Sonia C. Susano Chaves

¹

,

Diane Duroux

⁴

,

Ehsan Pishva

¹,

Diego Mastroeni

³,

Kristel Van Steen

^4,5,

Thomas Haaf

²

and

Daniël L. A. van den Hove

^1,6,*

¹

Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, 6229 ER Maastricht, The Netherlands

²

Institute of Human Genetics, Julius Maximilians University, 97074 Wuerzburg, Germany

³

Biodesign Institute, Neurodegenerative Disease Research Center, Arizona State University, Tempe, AZ 85281, USA

⁴

BIO3 Laboratory for Systems Genetics, GIGA-R Medical Genomics, University of Liège, 4000 Liège, Belgium

⁵

BIO3 Laboratory for Systems Medicine, Department of Human Genetics, KU Leuven, 3000 Leuven, Belgium

⁶

Laboratory of Translational Neuroscience, Department of Psychiatry, Psychosomatics and Psychotherapy, University of Wuerzburg, 97080 Wuerzburg, Germany

^*

Author to whom correspondence should be addressed.

Int. J. Mol. Sci. 2022, 23(24), 15571; https://doi.org/10.3390/ijms232415571

Submission received: 28 October 2022 / Revised: 28 November 2022 / Accepted: 29 November 2022 / Published: 8 December 2022

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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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.

Keywords: limiting dilution bisulfite pyrosequencing; laser-capture microdissection; DNA methylation; single-cell; epigenetics

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MDPI and ACS Style

Riemens, R.J.M.; Kenis, G.; Nolz, J.; Susano Chaves, S.C.; Duroux, D.; Pishva, E.; Mastroeni, D.; Van Steen, K.; Haaf, T.; van den Hove, D.L.A. Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP). Int. J. Mol. Sci. 2022, 23, 15571. https://doi.org/10.3390/ijms232415571

AMA Style

Riemens RJM, Kenis G, Nolz J, Susano Chaves SC, Duroux D, Pishva E, Mastroeni D, Van Steen K, Haaf T, van den Hove DLA. Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP). International Journal of Molecular Sciences. 2022; 23(24):15571. https://doi.org/10.3390/ijms232415571

Chicago/Turabian Style

Riemens, Renzo J. M., Gunter Kenis, Jennifer Nolz, Sonia C. Susano Chaves, Diane Duroux, Ehsan Pishva, Diego Mastroeni, Kristel Van Steen, Thomas Haaf, and Daniël L. A. van den Hove. 2022. "Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP)" International Journal of Molecular Sciences 23, no. 24: 15571. https://doi.org/10.3390/ijms232415571

APA Style

Riemens, R. J. M., Kenis, G., Nolz, J., Susano Chaves, S. C., Duroux, D., Pishva, E., Mastroeni, D., Van Steen, K., Haaf, T., & van den Hove, D. L. A. (2022). Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP). International Journal of Molecular Sciences, 23(24), 15571. https://doi.org/10.3390/ijms232415571

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

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