Post Mortem Tissue Flashcards
Hypothesis Driven Research
Begins with brain donation -
use PCR and ISH too look at the gene
-Look at protein using WB and IHC
-IHC – expression and location/cellularl/subcellular
-specific to antigen of interest
- direct ( AB labelled with fluorescent tag) and indirect (amplify signal) approach
Alzheimers and Astrocytes - Case study
Simpson JE et al
• Astrocytes play a role in maintaining homeostasis within the brain
• Dynamic cells that are actively involved in inflammatory & neurodegenerative events
-Secretion of cytokines & neurotoxic molecules
-Maintenance of extracellular environment
IHC
• GFAP- Main intermediate filament
• GFAP upreg in Reactive astrocytes
• EAAT2 (excitatory amino acid transporter-2)
EAAT2 main regulator of extracellular neurotransmitter glutamate
GFAP labelled astrocyte body
Different markers different patterns of staining
Case study Quantification of Expression
- 3 very distinct patterns of staining
- Computer software
- Take images from outer cortex in to the WM then use software
- Quantitative assessment – Analysis^D software (% area)
- Organized into Braak group
Case study Conclusion
- Gliosis & a change of astrocyte function is a common feature in the ageing population & partially correlates with Alzheimer-type pathology.
- Astrocyte response may relate to other factors independent of Alzheimer-type pathology.
- Case to case variation detected – error bars
Hypothesis Generating Research - overview
Microarray analysis of gene expression changers
Mass spectrometry analysis of protein changes
Hypothesis Generating Research
- Laser Capture Microdissection
- A number of studies of cellular identity have extrapolated from whole tissue investigations.
- Alternatively investigations have been carried out using tissue culture models.
- LCM allows the isolation of individual cells from their normal environment BUT untainted by other cell types.
- Issue with cell culture- isolation whereas in the brain the cells interact a lot with their surrounding
LCM
thermoplastic polymer transfer film
- Laser beam on the activated film tissue then slide on the tissue
Arcturus Pixcell system-applications
DNA sequencing DNA fingerprinting cDNA microarrays RT-PCR Proteomics Western blots
Arcturus Pixcell system- challenges
- Collect a pure cell population from heterogeneous tissue
- Obtain enough material for analysis
- Minimize sample loss during processing
- Maintain macromolecule integrity
- Reproducible analysis
- Laser may induce damage
Small sample preparation
- Preparation techniques designed for accurate identification of target cells
- Preservation of biologically relevant molecules for downstream analysis
- Dehydration required to inactivate nucleases and facilitate microdissection
Common Tissue Staining Techniques:
Colorimetric (H&E, Toluidine blue etc.)
Immunohistochemistry
In situ Hybridization (ISH)
Fluorescence
Staining must:
1) Facilitate cell identification
2) Preserve biomolecule integrity
3) Not inhibit LCM
How does LCM work
?
Absorbs near-IR energy
Distends predictably, evenly, reproducibly to enable selective targeting
Prevents laser energy from reaching sample
Becomes adhesive
Adhesion overcomes opposing forces to enable selective capture
LCM of motor Neurone
using Tolulin blue staining
Look at RNA quality
RNA samples run on Agilent Bioanalyser
Quality measured by presence of rRNA peaks & lack of degradation - Isolate RNA and run on Bioanalyser - Generates a RNA integrity no. - Quality measured but rRNA peaks Confirm enriched pop – PCR Look for NFL/GFAP/vWF/Beta-actin
Microarray analysis of LCM-ed cells.
using Affymetix gene chips
GeneChip Characteristics
• Each probe is an oligomer of 25 bases
• Probes come in pairs: perfect match (PM) and a mismatch (MM) probe
– The 13th nucleotide in the mismatch probe is non-complementary
– Mismatch probes control for hybridisation specificity
• Each transcript represented by 11 probe pairs
Issues of perfect match and mismatch of probes
On the chip – genome wide expression on one chip
Human Genome U133 Plus 2.0 Array
Genome wide expression on one microarray chip
Experimental design and considerations
Good experimental design is essential for achieving maximum information of statistical significance
-Number of biological replicates
3 replicates for low variability samples e.g. cell lines
5-6 replicates for animal models e.g. Tg mice
>10 replicates for high variability samples e.g. human tissue
On the chip - whole process
- mRNA extracted from cell
- Reverse transcriptase - cDNA
- Transcription - Biotin labeled cRNA
- Fragmentation - Fragmented biotin labeled cRNA
- Hybridize with Gene chip expression Array
- wash and stain
- Scan and Quantitate
Follow up preliminary Analysis
- Initial Analysis using Affymetrix software
- QC on hybridisation and scanning- Hybridisation controls checked
- Creates data table of hybridisation signals - All genes listed with calculated signal intensities
- Creates experimental report file - % genes present, marginal, absent
- Provides snap shot of all genes expressed in the sample when RNA was extracted
volcano plot
Volcano plots compare replicates of one sample v the other
Plots show fold change (one gene expressed more in one set than another) v probability that the result is significant , so grey are those genes changes less than 2 fold and not significant, purple are those not significantly altered, and orange those altered but not significant
Hierachical Clustering
Groups together samples with similar gene expression profiles
Analysis of all replicates
Characterisation of astrocyte gene expression changes in relation to AD pathology - Case study summary
Analysis shows downreg of signalling pathways – showing changes in insulin signalling down regulated in the astrocytes within the brain
-Gene expression profile of astrocytes with respect to increasing Braak stage reveals complex pattern of astrocyte dysfunction
Specific pathways affected include cell signalling, cytoskeleton & cell junctions
Impact on astrocyte survival & function including homeostasis
Astrocyte pathology may impair their neurotrophic function thereby contributing to the pathogenesis of Alzheimer-type pathology in the ageing brain
