Transcript Document

Microarrays,
RNAseq
And
Functional
Genomics
CPSC265
Matt Hudson
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
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
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
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
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.
Glass slide microarray summary
The process
Building the chip:
MASSIVE PCR
PCR PURIFICATION
and PREPARATION
PREPARING SLIDES
RNA
preparation:
CELL CULTURE
AND HARVEST
PRINTING
Hybing the
chip:
POST PROCESSING
ARRAY HYBRIDIZATION
RNA ISOLATION
DATA ANALYSIS
cDNA PRODUCTION
PROBE LABELING
steel
Robotically printed arrays
spotting pin
chemically modified slides
384 well source
plate
1 nanolitre spots
90-120 um diameter
Physical Spotting
Labelling RNA for Glass slides
Reverse Transcriptase
Reverse transcription
mRNA
Cy3 label
(control)
mRNA
(treated) Cy5 label
cDNA
Cy3 labelled
cDNA
Cy5 labelled
Hybridization
Binding of cDNA target samples to cDNA probes on the slide
Hybridize for
5-12 hours
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.
Hybridization chamber
3XSSC
HYB CHAMBER
ARRAY
LIFTERSLIP
SLIDE
LABEL
SLIDE LABEL
• Humidity
• Temperature
• Formamide
(Lowers the Tm)
Expression profiling with DNA microarrays
cDNA “B”
Cy3 labeled
cDNA “A”
Cy5 labeled
Laser 1
Hybridization
Laser 2
Scanning
+
Analysis
Image Capture
Image analysis
GenePix
Spotted cDNA microarrays
Advantages
• 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
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
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
Selection of Expression Probes
Set of oligos to be synthesized is defined, based on its
ability to hybridize to the target genes of interest
5’
3’
Sequence
Probes
Perfect Match
Mismatch
Chip
Computer algorithms are used to design photolithographic
masks for use in manufacturing
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
Affymetrix Wafer and Chip Format
20 - 50 µm
50… 11µm
Millions of identical
oligonucleotides
per feature
49 - 400
chips/wafer
1.28cm
up to ~ 400,000 “features” / chip
Labelling RNA for Affymetrix
Reverse Transcriptase
Reverse transcription
mRNA
cDNA
in vitro transcription
cRNA
Transcription
Biotin labelled
nucleotides
Target Preparation
B
Biotin-labeled
transcripts
B
B
B
B
Fragment
(heat, Mg2+)
B
B
cDNA
Fragmented cRNA
Wash & Stain
Scan
AAAA
mRNA
B
Hybridize
(16 hours)
®
GeneChip Expression Analysis
Hybridization and Staining
Array
Hybridized Array
cRNA Target
Streptravidinphycoerythrin
conjugate
Example:
Comparing
a mutant cell
line with a
wild type
line.
Instrumentation
Affymetrix GeneChip System
3000-7G Scanner
450 Fluidic Station
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.
Normal vs. Normal Normal vs. Tumor
Lung Tumor:
Up-Regulated
Lung Tumor:
Down-Regulated
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
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
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.
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
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
Protein separation:
2-dimensional gel electrophoresis
1st dimension
Separation by charge
(isoelectric focussing)
pI
pH 3
pH 10
2nd dimension
Separation by molecular weight
(SDS-PAGE)
kDa
Susan Liddel
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
150
100
75
50
37
25
20
Susan Liddel
Mass Spectrometry
FT-MS can tell you
10-20 residues of
sequence, but only
from a purified protein
Robots pick spots from
2-D gel, load into MS
Also, 2-D and 3-D LC
Array-based protein interaction detection
Protein microarrays
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 routinely
used to diagnose disease
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