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Brain research: Single neuron excision for RNA analysis

Improve your workflow with laser microdissection

Parkinson’s disease is a common progressive neurodegenerative disorder. It is connected with cell death of dopamine-releasing neurons in the substantia nigra. Differences in gene expression patterns between individual dopamine-releasing neurons of disease affected and healthy individuals allow defining target genes for therapies. For RNA analysis, single cell resolution is crucial, as investigating the whole tissue is meaningless. Analyzing mixtures of dopamine-releasing neurons and all other brain cells distorts the result.

Leica’s laser microdissection (LMD) method allows you to specifically isolate and analyze individual dopaminergic neurons from post mortem tissues of Parkinson’s disease patients. This allows the researcher to exclude misinterpretations due to the examination of inhomogeneous cell populations.

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Please contact us for a demo request or personal expert advice on our microscopy solutions for RNA analysis.

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Leica LMD6 & LMD7

Leica LMD6 & LMD7

Laser Microdissection enables users to isolate specific single cells or entire areas of tissue.

RNA Analysis Workflow with Single Cell Precision

Leica's Laser Microdissection systems will improve your workflow by precisely cutting only the cells you are interested in. Everything under visual control and without contamination with surrounding tissue.

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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 brings the tissue sample into a constitution which can be cut 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 cryo-sectioned subsequently.

We will deliver the suitable LMD slides for your application.

2. Fixation and staining

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.

Beside Cresyl violet other stains (e.g. toluidine blue) can be used as well.

3. Visualization and ROI

The Leica LMD systems can generate an automatic sample overview for you that can be used to easily navigate to your regions of interest (ROI). 

Your ROI e.g. Dopaminergic neurons, can be identified and marked automatically by the AVC 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 contaminations e.g. with RNases. Also, you can cut your samples direct on the fly with the "Move and Cut" tool without any predefined shapes. The collection via gravity allows to use standard, cost-effective consumables such as PCR tubes or 8-strip tubes caps. The collection can be done directly into lysis buffer or dry. We provide the appropriate LMD system according to 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, quantitative, real-time PCR, or can be stored at –20°C until needed.

Please refer to www.qiagen.com for details.

6. RNA analysis

RNA analysis is classically done via qPCR. Special LMD protocols will ensure that the RNA quality remains untouched.

Technologies such as Microarrays or next generation sequencing (NGS) are applicable as well.

Thanks to the LMD cutting precision you will get results only of the cells you are interested in. This enables you to compare gene expression results of different tissue areas reliably.

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Typical fields of research

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

References

  • Duda et al. Laser Capture Microdissection (2018) pp 335-360
  • Lee et al. CellRep (2017)
  • Green et al. PNAS (2017)
  • Ortner et al. NerobioDis (2017)