Post Mortem Tissue

Question 1

Hypothesis Driven Research

Answer 1

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

Question 2

Alzheimers and Astrocytes - Case study

Simpson JE et al

Answer 2

• 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

Question 3

Case study Quantification of Expression

Answer 3

• 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

Question 4

Case study Conclusion

Answer 4

• 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

Question 5

Hypothesis Generating Research - overview

Answer 5

Microarray analysis of gene expression changers

Mass spectrometry analysis of protein changes

Question 6

Hypothesis Generating Research

Answer 6

• 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

Question 7

LCM

Answer 7

thermoplastic polymer transfer film
- Laser beam on the activated film tissue then slide on the tissue

Question 8

Arcturus Pixcell system-applications

Answer 8

DNA sequencing
DNA fingerprinting
cDNA microarrays
RT-PCR
Proteomics
Western blots

Question 9

Arcturus Pixcell system- challenges

Answer 9

• 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

Question 10

Small sample preparation

Answer 10

• 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

Question 11

Common Tissue Staining Techniques:

Answer 11

Colorimetric (H&E, Toluidine blue etc.)
Immunohistochemistry
In situ Hybridization (ISH)
Fluorescence

Question 12

Staining must:

Answer 12

1) Facilitate cell identification
2) Preserve biomolecule integrity
3) Not inhibit LCM

Question 13

How does LCM work

?

Answer 13

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

Question 14

LCM of motor Neurone

Answer 14

using Tolulin blue staining

Question 15

Look at RNA quality

Answer 15

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

Question 16

Microarray analysis of LCM-ed cells.

Answer 16

using Affymetix gene chips

Question 17

GeneChip Characteristics

Answer 17

• 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

Question 18

Human Genome U133 Plus 2.0 Array

Answer 18

Genome wide expression on one microarray chip

Question 19

Experimental design and considerations

Answer 19

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

Question 20

On the chip - whole process

Answer 20

1. mRNA extracted from cell
   - Reverse transcriptase
2. cDNA
   - Transcription
3. Biotin labeled cRNA
   - Fragmentation
4. Fragmented biotin labeled cRNA
5. Hybridize with Gene chip expression Array
   - wash and stain
   - Scan and Quantitate

Question 21

Follow up preliminary Analysis

Answer 21

• 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

Question 22

volcano plot

Answer 22

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

Question 23

Hierachical Clustering

Answer 23

Groups together samples with similar gene expression profiles
Analysis of all replicates

Question 24

Characterisation of astrocyte gene expression changes in relation to AD pathology - Case study summary

Answer 24

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