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Improving RNA Analysis with Laser Microdissection

Brain research: Single neuron excision for a better RNA-analysis workflow

Parkinson’s disease is a progressive neurodegenerative disorder connected with cell death of dopamine-releasing neurons in the brain. Differences in gene expression between individual dopamine-releasing neurons of disease-afflicted and healthy individuals allow target genes to be defined. For RNA analysis, single-cell resolution is crucial. Analyzing mixtures of dopamine-releasing neurons and other cells distorts the results. Single  dopaminergic neurons from tissues can be isolated and analyzed with laser microdissection (LMD). Misinterpretations due to inhomogeneous cell populations are excluded.

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RNA Analysis Workflow with Single Cell Precision

Laser microdissection (LMD) systems from Leica Microsystems can improve your workflow by enabling you to precisely cut only the cells you are interested  in. Everything remains under visual control  and without contamination  by surrounding tissue.

RNA Analysis Workflow - Steps

1. Sample preparation

Typically, special membrane-based slides are used for laser microdissection. Sample preparation  for LMD is straightforward and can be derived from classical preparations for histology. Paraffin embedding gives the tissue sample a better consistency for cutting it into thin slices. Alternatively, you can utilize cryo-sectioning to prepare your slides. Tissue for RNA analysis is often flash-frozen, e.g., in liquid nitrogen, and then cryo-sectioned.

2. Fixation and staining

The cresyl violet stain is commonly used for neuronal tissue. It is a basic stain that binds to acidic molecules of neuronal cytoplasm, such as, RNA-rich ribosomes. Cresyl violet permanently stains a section and is suitable for both paraffin and frozen sections and subsequent RNA preparation. Besides cresyl violet, other stains like toluidine blue can be used as well.

3. Visualization and ROI

Leica LMD systems can generate an automatic sample overview  that helps you easily navigate to regions of interest (ROIs).
Your ROI, e.g., dopaminergic neurons, can be identified and marked automatically by the ADM  software module or you can apply shapes manually.

4. Laser microdissection

Next, the defined ROIs are precisely  dissected by the laser under visual control and directly collected via gravity. Contact-free excision helps prevent contamination, e.g., with RNases. Also, you can cut your samples directly on the fly with the "Move and Cut"  tool without any predefined shapes. The collection via gravity allows you to use standard, cost-effective  consumables, such as PCR tubes or 8-strip tube caps. The collection can be done directly into a lysis buffer solution or dry air.

Leica Microsystems can provide you with the LMD system which is appropriate  for your needs.

5. Extraction of RNA

Leica Microsystems recommends the high-quality QIAGEN kits (RNeasy® Micro Kit) for preparation of nucleic acids. They can be immediately used in downstream applications, such as PCR, sequencing, and quantitative and real-time PCR, or they can be stored at –20°C until needed at a later time. Please refer to www.qiagen.com for details.

6. RNA analysis

RNA analysis is classically done via qPCR. Special LMD protocols ensure that the RNA quality remains untouched. Technologies like microarrays or next generation sequencing (NGS) are applicable as well. Thanks to the LMD cutting precision, you will get results only for the cells you are interested in. This capability enables you to compare gene expression results of different tissue areas reliably.

Typical fields of research

  • Alzheimer's disease
  • Developmental biology
  • Aging
  • Mutation analysis of tumor tissue
  • Other diseases

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