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A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones
Published on: September 16, 2019
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A method for isolating high quality RNA from mouse cortical and cancellous bone.
Natalie H Kelly1, John C Schimenti2, F Patrick Ross3
1Sibley School of Mechanical and Aerospace Engineering, Cornell University, 105 Upson Hall, Ithaca, NY 14853, USA; Department of Biomedical Engineering, Cornell University, 101 Weill Hall, Ithaca, NY 14853, USA.
Bone
|July 30, 2014
Summary
This study developed a method to isolate RNA from mouse bone, distinguishing between cortical and cancellous bone. This technique accurately measures gene expression, crucial for understanding bone diseases and fragility fractures.
Area of Science:
- Molecular Biology
- Bone Biology
- Genomics
Background:
- Fragility fractures highlight the need to study gene expression in distinct bone compartments (cortical and cancellous).
- Marrow contamination can confound gene expression measurements in bone tissue.
- Site-specific differences in bone response to load and disease necessitate separate analyses.
Purpose of the Study:
- To develop and validate a method for isolating RNA from mouse cortical and cancellous bone.
- To assess the efficiency of marrow removal techniques (flushing vs. centrifugation) on RNA quality and gene expression.
- To investigate site-specific gene expression differences between cortical and cancellous bone.
Main Methods:
- RNA isolation from intact and marrow-removed mouse tibiae (cortical and cancellous bone).
- Comparison of marrow removal methods: flushing versus centrifugation.
- Gene expression analysis of bone-related genes (Col1a1, Bglap) and an erythroid marker (Icam4).
Main Results:
- Centrifugation was more effective than flushing in removing marrow cells, indicated by histology and reduced Icam4 expression.
- Marrow-free bone samples showed significantly higher expression of Col1a1 and Bglap compared to samples with intact marrow.
- Cortical bone exhibited higher expression of Col1a1 and Bglap than cancellous bone.
Conclusions:
- A novel RNA isolation approach enables accurate, site-specific gene expression analysis in cortical and cancellous bone.
- Effective marrow removal is critical for reliable gene expression studies in bone.
- This method facilitates the study of skeletal site-specific changes relevant to bone health and disease.
