microarrays, rnaseq and functional genomics cpsc265 matt hudson
TRANSCRIPT
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Microarrays,RNAseq AndFunctionalGenomics
CPSC265
Matt Hudson
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Microarray Technology• Relatively young technology – • Already mostly obsolete, though.
• Usually used like a Northern blot – can determine the amount of mRNA for a particular gene
• Except – a Northern blot measures one gene at a time
• A microarray can measure every gene in the genome, simultaneously
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Recent! History
• 1994. First microarrays developed by Ron Davis and Pat Brown at Stanford.
• 1997-1999. Practical microarrays become available for yeast, humans and plants
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Why analyze so many genes?
• Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function.
• To find genes involved in a particular process, we can look for mRNAs “up-regulated” during that process.
• For example, we can look at genes up-regulated in human cells in response to cancer-causing mutations, or look at genes in a crop plant responding to drought.
• Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways
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Two Main Types of Microarray
Oligonucleotide, photolithographic arrays“Gene Chips”
Miniaturized, high density arrays of oligos (Affymetrix Inc., Nimblegen, Inc.)
Printed cDNA or Oligonucleotide Arrays Robotically spotted cDNAs or Oligonucleotides • Printed on Nylon, Plastic or Glass surface• Can be made in any lab with a robot• Several robots in ERML• Can also buy printed arrays commercially
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The original idea
A microarray of thousands of genes on a glass slide
Each “spot” is one gene, like a probe in a Northern blot.
This time, the probes are fixed, and the target genes
move about.
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Glass slide microarray summary
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The processBuilding the chip:
MASSIVE PCR PCR PURIFICATION and PREPARATION
PREPARING SLIDES PRINTING
RNA preparation:
CELL CULTURE AND HARVEST
RNA ISOLATION
cDNA PRODUCTION
Hybing the chip: POST PROCESSING
ARRAY HYBRIDIZATION
PROBE LABELING
DATA ANALYSIS
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Robotically printed arraysRobotically printed arrays
1 nanolitre spots90-120 um diameter
384 well source plate
chemically modified slides
steel
spotting pin
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Physical Spotting
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Reverse Transcriptase
Labelling RNA for Glass slides
mRNA(control)
cDNACy3 labelled
Reverse transcription
mRNA(treated)
cDNACy5 labelled
Cy3 label
Cy5 label
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HybridizationBinding of cDNA target samples to cDNA probes on the slide
cover
slip
Hybridize for
5-12 hours
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Northern blot vs. Microarray• In Northern blotting, the whole mRNA of the
organism is on the membrane. The labelled “probe” lights up a band – one gene
• In a microarray, the whole genome is printed on a slide, one “probe” spot per gene. Mixed, labelled cDNA, made from mRNA from the organism, is added. Each probe lights up green or red according to whether it is more or less abundant between the control and the treated mRNA.
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LABEL
3XSSC
HYB CHAMBER
ARRAY
SLIDE
LIFTERSLIP
SLIDE LABEL
• Humidity• Temperature• Formamide
(Lowers the Tm)
Hybridization chamber
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Expression profiling with DNA microarrays
cDNA “A ”Cy5 labeled
cDNA “B”Cy3 labeled
Hybridization Scanning
Laser 1 Laser 2
+
Analysis Image Capture
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Image analysis
GenePix
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Spotted cDNA microarraysAdvantages• Lower price and flexibility• Can be printed in well equipped lab• Simultaneous comparison of two related
biological samples (tumor versus normal, treated versus untreated cells)
Disadvantages• Needs sequence verification• Measures the relative level of expression
between 2 samples
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Affymetrix Microarrays
• One chip per sample• Made by photolithography• ~500,000 25 base probes
…unlike Glass Slide Microarrays
•Made by a spotting robot•~30,000 50-500 base probes•Involves two dyes/one chip•Control and experiment on same chip
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Affymetrix GeneChip
Miniaturized, high density arrays of oligos 1.28-cm by 1.28-cm (409,000 oligos)
Manufacturing Process
Solid-phase chemical synthesis and Photolithographic fabrication techniques employed in semiconductor industry
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Selection of Expression ProbesSet of oligos to be synthesized is defined, based on its ability to hybridize to the target genes of interest
Probes
Sequence
Perfect Match
MismatchChip
5’ 3’
Computer algorithms are used to design photolithographic masks for use in manufacturing
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Photolithographic Synthesis
Manufacturing Process
Probe arrays are manufactured by light-directed chemical synthesis process which enables the synthesis of hundreds of thousands of discrete compounds in precise locations
Lamp
Mask Chip
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Affymetrix Wafer and Chip Format
1.28cm
50… 11µm
20 - 50 µm
Millions of identical oligonucleotides
per feature
49 - 400 chips/wafer
up to ~ 400,000 “features” / chip
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Reverse Transcriptase
in vitro transcription
Labelling RNA for Affymetrix
mRNA cDNA
Reverse transcription
TranscriptionBiotin labellednucleotidescRNA
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Target Preparation
cDNA
Wash & Stain
Scan
Hybridize
(16 hours)
mRNAAAAA
B B B B
Biotin-labeled transcripts Fragment
(heat, Mg2+)
Fragmented cRNA
B B
B
B
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GeneChip® Expression Analysis
Hybridization and Staining
Array
cRNA Target
Hybridized Array
Streptravidin-phycoerythrinconjugate
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Example:
Comparing a mutant cellline with awild typeline.
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Instrumentation
Affymetrix GeneChip System3000-7G Scanner
450 Fluidic Station
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Microarray data analysis
This is now a very important branch of statistics
It is unusual to do thousands of experiments at once. Statistical methods didn’t exist to analyse microarrays. Now they are being rapidly developed.
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Normal vs. Normal Normal vs. Tumor
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Lung Tumor: Up-Regulated
Lung Tumor: Down-Regulated
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Microarray Technology - Applications• Gene Discovery-
– Assigning function to sequence– Finding genes involved in a particular process– Discovery of disease genes and drug targets
• Genotyping– SNPs – Genetic mapping (Humans, plants)– Patient stratification (pharmacogenomics)– Adverse drug effects (ADE)
• Microbial ID
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Why it is becoming obsolete
• In a word, RNAseq
• RNAseq uses DNA sequencing to do the same thing.
• Rather than an array, you just sequence millions of mRNA fragments, then figure out what genes they are from
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Why RNAseq only just caught on
• It’s been around for a long time, called things like SAGE and MPSS.
• But they were expensive and arrays were cheap. Now, sequencing is as cheap as arrays
• Also, you need a fully sequenced reference genome for the computer analysis.
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What RNAseq / arrays can’t do
• Tell you anything about protein levels
• Tell you anything about post-translational modification of proteins
• Tell you anything about the structure of proteins
• Predict the phenotype of a genetic mutant
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Proteomics
• A high througput approach to learning about all the proteins in a cell
• As microarrays are to a Northern blot, proteomics is to a Western blot
• Two main approaches – • 2D gels + MS• Protein microarrays
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Protein separation: 2-dimensional gel electrophoresis
1st dimension Separation by charge(isoelectric focussing)
2nd dimension Separation by molecular weight
(SDS-PAGE)kDa
pH 3 pH 10
pI
Susan Liddel
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Proteins extracted from cow ovarian follicle granulosa cells separated on a broad range IPG strip (pH3-10)
followed by a 12.5% polyacrylamide gel, silver stained
3.5 9.0
20
150
100
75
50
37
25
Susan Liddel
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Mass Spectrometry
FT-MS can tell you10-20 residues ofsequence, but onlyfrom a purified protein
Robots pick spots from2-D gel, load into MS
Also, 2-D and 3-D LC
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Array-based protein interaction detection
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Protein microarrays
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The future of microarrays:
•Still looking good, in areas other than research
•Used by pharmaceutical companies, medical diagnostics, etc.
•In the future, just like silicon chips, likely to get cheaper, faster and more powerful
•It may not be long before they are routinelyused to diagnose disease
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The future of proteomics:
• Many people will tell you proteomics IS the future of biology
• If they can get it to work as well as microarrays, they will be right
• The problem is, every protein has different chemistry, while all mRNAs are closely comparable
• At the moment, proteomics is a hot field, but few major biological discoveries have been made with proteomics – many have been made with microarrays