20/04/12
What is a Microarray?
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Microarray has become a general term, there are many types now
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Microarray practical
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DNA microarrays Protein microarrays Transfection microarrays Tissue microarray
Well be discussing DNA microarrays
Louise Lutze-Mann
What is a DNA Microarray?
A grid of DNA spots (probes) on a support used to detect complementary sequences } The DNA spots can be deposited by
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DNA microarrays
Piezolectric (ink jet style) Pen Photolithography (Affymetrix)
The support can be plastic, glass, silicon (Affymetrix) RNA/DNA of interest is labelled and hybridizes with the array } Hybridization with probes is detected optically.
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Global survey of gene expression
cDNA Arrays
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Types of DNA microarrays
What is measured depends on the chip design and the laboratory protocol: } Expression
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Expression arrays for breast cancer
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Resequencing Tiling
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Measure mRNA expression levels (usually polyadenylated mRNA) Detect changes in genomic regions of interest
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SNP ?...
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Tiles probes over an entire genome for various applications (novel transcripts, ChIP, epigenetic modifications) Detect which known SNPs are in the tested DNA
295 primary breast cancers Women under 53 years of age 70 genes found to be prognostic
Kaplan-Meier plot for 151 breast cancer patients followed for 10 years
What do Expression Arrays really measure? Gene Expression mRNA levels in a cell } mRNA levels averaged over a population of cells in a sample } relative mRNA levels averaged over populations of cells in multiple samples } relative mRNA hybridization readings averaged over populations of cells in multiple samples } some relative mRNA hybridization readings averaged over populations of cells in multiple samples
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Patients with a good signature derive no benefit from adjuvant chemotherapy
It appears that some transcripts just cant be detected accurately by these techniques.
Central Assumption of Gene Expression Microarrays
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The project
Patients with schizophrenia have a reduced cancer incidence compared with the rest of the population } We found that the drugs that are used to treat schizophrenia inhibit the growth of cancer cells
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The level of a given mRNA is positively correlated with the expression of the associated protein.
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Higher mRNA levels mean higher protein expression, lower mRNA means lower protein expression Protein degradation, mRNA degradation, polyadenylation, codon preference, translation rates, alternative splicing, translation lag
Other factors:
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This is relatively obvious, but worth emphasizing
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Antipsychotic drugs affect the growth of breast cancer cells
The project
How? Why? We found is that these drugs impact cholesterol synthesis and transport in the cell } Cholesterol is required to synthesis membranes in new cells } Statins are drugs that block HMG-CoA reductase the first committed step in cholesterol synthesis } These drugs are currently in use to treat hypercholesterolaemia
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Response to a statin varies in the different breast cancer cell lines WHY?
Why the difference in response to statin?
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Two cell lines:
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MB-175 sensitive SK-BR-3 relatively resistant
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Treated with 10 M mevastatin for 24 hrs RNA was isolated and used to make cDNA Microarray analysis Genes that are differentially regulated will be identified Need to confirm by RT-PCR YOU!!!!!!
Affymetrix Expression Arrays
http://www.affymetrix.com/technology/ge_analysis/index.affx
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Data generated:
Gene identity SKBR-3 treated cells vs control MB175 treated cells vs control MB175 control vs SKBR-3 control MB175 treated vs SKBR-3 treated
Venn diagram showing genes shared between cell lines and/or treatment
3449 lines of data
Prac Class
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Prac class - Semi-quantitative RT-PCR
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Week 1
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Microarray on-line virtual experiment Tour of Ramaciotti Centre Isolate RNA Analysis of microarray results Reverse transcription of mRNA and PCR on cDNA Analysis and report writing
Week 2
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Treat cells (done for you) Harvest cells and isolate RNA using Trizol 2 cell lines
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MB-175 control and drug-treated SK-BR-3 control and drug-treated
Week 3
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Week 4
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Each pair of students will isolate RNA from one cell line, so 2 samples (control and treated) } Reverse transcribe mRNA (how?) } PCR with gene-specific primers which genes? } Analyse on gel
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Purity & Quantity of Sample
Nucleic Acid Quality Control
Dr. Helen Speirs
NanoDrop ND-1000
Need only 1 l of sample. No cuvette required. Broad spectral output. Detection limit 2 3000ng for RNA
Sample Quality
Purity & Quantity
Quality/Integrity
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The Good Pure RNA
The Bad
The Bad Made Good
The Ugly - Trizol Contamination
clean
original
Intact RNA - eukaryotic
28S rRNA 18S rRNA
Assessing RNA Quality - Agilent Bioanalyzer Service
28S rRNA 18S rRNA
marker 5S region
28S rRNA 18S rRNA
28S rRNA 18S rRNA
5S rRNA
5S region
5S region
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Partially Degraded RNA
Bacterial Contamination in Cultured Cells
28S marker 18S 23S
16S
