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Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses
Chih-Hsiang Yang1, John Longinotto2, Ilaria Panzeri1
1Van Andel Institute; Max Planck Institute of Immunobiology and Epigenetics.
Journal of Visualized Experiments : Jove
|July 5, 2022
Summary
This study presents an optimized method for isolating mature adipocytes, crucial for understanding obesity. The new protocol effectively removes lipids, improving RNA and chromatin quality for genetic studies.
Area of Science:
- Cell Biology
- Genetics
- Epigenetics
Background:
- Obesity is a complex multifactorial disease.
- Mature adipocytes are key cells in white adipose tissue, vital for understanding obesity's origins.
- Existing methods for isolating adipocyte RNA and chromatin face challenges due to high lipid content.
Purpose of the Study:
- To develop an optimized protocol for isolating mature murine adipocytes.
- To improve the yield and quality of RNA and chromatin from adipocytes for downstream analyses.
- To facilitate whole-genome transcriptome and epigenome studies related to obesity.
Main Methods:
- Utilized a novel procedure involving heptane for efficient lipid separation from adipocytes.
- Optimized the isolation of mature adipocytes from murine adipose tissue.
- Validated the protocol for subsequent RNA and chromatin analysis.
Main Results:
- The optimized protocol yields high-integrity RNA, suitable for high-quality RNA-sequencing (RNA-seq).
- The procedure enhances nuclear isolation yield, producing reproducible chromatin immunoprecipitation-sequencing (ChIP-seq) results.
- Demonstrated a reliable and universal method for murine adipocyte isolation.
Conclusions:
- This protocol offers a reliable and universal approach for isolating murine adipocytes.
- The method is suitable for comprehensive whole-genome transcriptome and epigenome studies.
- Improved adipocyte isolation is essential for advancing obesity research.
