研究目的
To develop an LCM protocol that yields high-quality RNA from fresh-frozen mouse bones for gene expression analysis.
研究成果
The developed LCM protocol successfully obtained sufficient quantity of high-quality RNA from fresh-frozen mouse bones, suitable for gene expression analysis. The protocol can be adapted for studying in situ gene expression in cells of any hard tissue under various conditions.
研究不足
The protocol was optimized particularly for the LCM system that uses gravity for sample collection and may not be directly applicable to other LCM systems without adjustments.
1:Experimental Design and Method Selection:
The study evaluated the effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system on RNA yield and integrity.
2:Sample Selection and Data Sources:
Bone tissues from 3-month-old male wild type C57BL/6 mice were used.
3:List of Experimental Equipment and Materials:
Included adhesive film, commercial LCM frozen section stain, PET membrane, LCM system, and column-based RNA extraction kit.
4:Experimental Procedures and Operational Workflow:
Involved freeze embedding, section preparation, rapid staining protocol, laser capture microdissection, RNA extraction, and measurement of RNA yield and integrity.
5:Data Analysis Methods:
RNA yield and integrity were measured using micro-capillary electrophoresis.
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