A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

Summary

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

Abstract

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-µm-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

Introduction

Tissues are made up of heterogeneous and spatially distributed cell types. Different cell types in a given tissue may respond differently to the same signal. Therefore, it is essential being able to isolate specific cell populations for the assessment of the role of different cell types in both physiological and pathological conditions. Laser capture microdissection (LCM) offers a relatively rapid and precise method for isolating and removing specified cells from complex tissues¹. LCM systems use the power of a laser beam to separate the cells of interest from histological tissue sections without the need for enzymatic processing or growth in c....

Protocol

Bone tissue from mice was used in strict accordance with prevailing guidelines for animal care and all efforts were made to minimize animal suffering.

1. Animals and Freeze Embedding

1. House animals in conditions of constant room temperature (RT; 24 °C) and a 12 h light/12 h dark cycle with free access to food and water.
   NOTE: Bone tissues in this study were obtained from 3-month-old male wild type C57BL/6 mice.
2. At necropsy, exsanguinate mice from the abdominal vena cava under general anesthesia (ketamine/xylazine, 100/6 mg/kg intraperitoneal).
   NOTE: In order to avoid ....

Results

An LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells of mouse femurs. In the optimized protocol, 8 µm-thick frozen bone sections were cut on an adhesive film and stained using a rapid protocol for a commercial LCM frozen section stain. The sample was sandwiched between the PET membrane and the adhesive film. Mouse bone cells were microdissected using an LCM system that uses gravity for sample collection. A column-based RNA extraction method was used to.......

Discussion

Both RNA quality and quantity can be affected negatively at all stages of the sample preparation such as tissue manipulation, LCM process, and RNA extraction. Therefore, an LCM protocol was developed to obtain sufficient amount of high-quality RNA for subsequent gene expression analysis.

For LCM, most laboratories use sections 7−8 µm thick². Thicker sections would allow more material to be harvested. However, if they are too thick, this could reduce the microscopic resolu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Sigma	63689-25ML-F
Absolute ethanol EMPLURA	Merck Millipore	8,18,76,01,000
Adhesive film (LMD film)	Section-Lab	C-FL001
Agilent 2100 Bioanalyzer System	Agilent Technologies
Agilent RNA 6000 Pico Chip Kit	Agilent Technologies	5067-1513
Arcturus HistoGene Staining Solution	Applied Biosystems	12241-05
Cryofilm fitting tool	Section-Lab	C-FT000
Cryostat Leica CM 1950	Leica Biosystems
Glass microscope slides, cut color frosted orange	VWR Life Science	631-1559
Histology tissue molds PVC	MEDITE	48-6302-00
LMD7 Laser Mikrodissektion System	Leica Microsystems
Low profile Microtome Blades Leica DB80 XL	Leica Biosystems	14035843496
Nuclease-free water	VWR Life Science	E476-500ML
PET membrane slides 1.4 μm	Molecular Machines & Industries GmbH	50102
RNase Away surface decontaminant	Molecular BioProduct	7002
RNeasy Micro Kit	Qiagen	74004
Tissue-Tek optimal cutting temperature (OCT) compound	Sakura Finetek	4583
Xylene	VWR Life Science	2,89,73,363