Download - Department of Molecular Biology and Genetics, Faculty

Introduction to Microarrays
Alexander W. Bruce.
[email protected]
Lecture theatre C3, Faculty of Science
Download lecture slides from the Department of Molecular Biology’s website, under study
materials, 2015 Summer Microscopy Workshop (temporary address – see David Dolezel):
http://kmb.prf.jcu.cz/en/en-materials/study-materials/2015-summer-microscopy-workshop
Advanced methods in molecular biology - Introduction to microarrays
Topics to be covered
• Introduction and history of the microarray
• Types of microarray
• Microarray applications (plus case study)
• Advanced DNA sequencing and the future of
microarrays . . . . .
Not necessarily in that order . . .
Advanced methods in molecular biology - Introduction to microarrays
Central dogma
GENOME
TRANSCRIPTOME
PROTEOME
DNA/ genes
RNA/ transcripts
protein
How can we measure components of the TRANSCRIPTOME i.e. transcripts?
Advanced methods in molecular biology - Introduction to microarrays
Nucleic acid hybridisation
Duplex of
complementary basepairing strands
Denaturation of
strands e.g. salts and
heat
Hybridisation of
complementary
strands e.g. labeled
probes
Advanced methods in molecular biology - Introduction to microarrays
Southern blotting (DNA)
Northern blotting (RNA)
Detects one or two specific nucleic acid
sequences in a complex sample/mixture
Advanced methods in molecular biology - Introduction to microarrays
How can multiple specific
nucleic acids (e.g. mRNAs) be
detected in a sample at the
same time ?
Answer: invert the system i.e. immobilise the
probes and label the sample
Advanced methods in molecular biology - Introduction to microarrays
Cancer Res. 1982 Mar;42(3):1088-93.
Cloning and screening of sequences expressed in a mouse colon tumor.
Augenlicht LH, Kobrin D.
PMID: 7059971 [PubMed - indexed for MEDLINE] Free Article
Lysed bacterial clones with plasmid vectors containing known specific cDNA sequences
(378), spotted as arrays and replica plated onto nylon membranes and hybridised with
radioactively labeled sample cDNA
The ancestral microarray !?
Advanced methods in molecular biology - Introduction to microarrays
Cancer Res. 1987 Nov 15;47(22):6017-21.
Expression of cloned sequences in biopsies of human colonic tissue and in
colonic carcinoma cells induced to differentiate in vitro.
Augenlicht LH, Wahrman MZ, Halsey H, Anderson L, Taylor J, Lipkin M.
PMID: 3664505 [PubMed - indexed for MEDLINE] Free Article
Method improved and expanded to include 4000 cDNA bacterial clones of human cDNAs
automatically spotted as liquid cultures onto membranes before lysis.
Some commercial exploitation of this semi-quantitative technique e.g. ‘Clonetech
expression blots’
Advanced methods in molecular biology - Introduction to microarrays
Arraying PCR products
PCR amplifying cDNA from plasmid cDNA library collections
Patrick Brown
cDNAs
synthesised
from mRNA
Cloning
into a
common
plasmid
vector
Organised cDNA library
PCR amplify specific
cDNAs from each
well with the same
primer pairs and spot
on microarray
Single colony
innoculation into
multiwell plates
Transform
bacteria
cDNA library
Advanced methods in molecular biology - Introduction to microarrays
The first ‘true’ microarray
Science. 1995 Oct 20;270(5235):467-70.
Quantitative monitoring of gene expression patterns with a complementary DNA
microarray.
Schena M, Shalon D, Davis RW, Brown PO.
PMID: 7569999 [PubMed - indexed for MEDLINE]
Automated robotic manufacture
Direct ‘spotting’ of purified
cDNA (ds) onto coated glass
slides
High density arrays of
cDNA probes on
microscope slides i.e. each
spot comprises one
specific cDNA sequence
Quantitative
‘Microarrays’
Advanced methods in molecular biology - Introduction to microarrays
COMPETITIVE HYBRIDISATIONS
Experimental & Reference samples
Purified RNA
cDNA synthesis incorporating different
fluorescently labeled dNTPs for
experimental and reference RNAs
e.g.Cy3 or Cy5 dyes
The ratio of the
emitted fluorescence
for each labeled
sample and per spot/
probe permits the
deduction of the
relative fold change in
mRNA expression
between the samples
for the gene
represented by that
spot.
Co-hybridisation of differently labeled sample cDNAs to
microarray of double stranded cDNA probes, washing and
laser excitation of each fluorophore and recording of
emitted flourescence
Advanced methods in molecular biology - Introduction to microarrays
GENOME-WIDE MICROARRAY
Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):13057-62.
Yeast microarrays for genome wide parallel genetic and gene expression analysis.
Lashkari DA, DeRisi JL, McCusker JH, Namath AF, Gentile C, Hwang SY, Brown PO, Davis RW.
Source
Department of Genetics, Stanford University, CA 94305, USA.
PMID: 9371799
An array of cDNAs from all known ORFs in the yeast
genome (yeast genome DNA sequence had been
released in April 1996)
Comparison of mRNA expression between yeast
growing in either glucose or galactose contatining
media
See red and green spots for relative expression
differences and note most spots are yellow because
there is no change in expression between the two
samples
Interrogation of the ENTIRE TRANSCRIPTOME in a single experiments
Advanced methods in molecular biology - Introduction to microarrays
Refinements of microarray based
techniques
Sample cDNA fluorescent labeling methods
Cy3/5
Cy3/ Cy5 coupled dNTPs
Amino-allyl modified dNTPs are more efficiently
incorporated into cDNA . . .
Poor rates of incorporation by reverse
transcriptase enzymes due to ‘bulky’ Cy dye
moiety
. . . and the fluorescent Cy dyes can then be
coupled to this reactive amine after completion of
cDNA synthesis
Advanced methods in molecular biology - Introduction to microarrays
Refinements of microarray based
techniques
Array manufacture
• traditionally double stranded (ds)
Probe cDNAs generated by PCR where
one of the oligo primers used has a 5’
amino modification
cDNAs were arrayed
• glass slides would be coated (e.g.
poly-L-lysine) to facilitate ds cDNA
binding
• REDUCED SENSITIVITY because the
two strands could self anneal
therefore blocking the hybridization
of the labeled sample cDNA
• SINGLE STRANDED microarrays
developed
A
A
A
A
A
cDNAs then spotted on coated slides and
covalently attached via amino group.
Denaturing the spotted DNA leaves a
single strand available for highly efficient
hybridisation with the labeled sample
Advanced methods in molecular biology - Introduction to microarrays
Spotted oligonucleotide arrays
Publication of genome sequences permit the
design of ‘short’ DNA oligonucleotides (25 70 nts) that are sequencfe specific for
particular genes.
These single stranded oligos can then by
spotted onto the microarray slide and
hybridised in ‘ two colour competitive
hybridisations’
Oligo probes
Advanced methods in molecular biology - Introduction to microarrays
Spotted oligonucleotide microarrays
• The probe features on the microarray are
deposited onto the slide as presynthesised/
complete entities (e.g. orignally as bacterial clones
and lately as cDNA PCR products or oligos)
• They are usually used in ‘two colour competitive
hybridisations’ involving differently labled
‘experimental’ and ‘reference’ samples
• They can be relatively easily made ‘in house’ and
as time has progressed they have become cheaper
Although they were the first ‘true’ microarrays
they are not the only type available!
Advanced methods in molecular biology - Introduction to microarrays
In situ synthesised oligonucleotide
arrays
e.g. ‘Affymetrix GeneChip’
• single stranded oligonucleotides
(25 nts for Affy) are directly
synthesised onto the array surface
on nucleotide at a time (e.g.
photochemical lithography)
• permits a very high density of
probes on a single microarray (e.g. all
known ORF human genome)
• highly commercialised and highly
standardised protocols
• highly SENSITIVE and SPECIFIC
when compared to traditional
spotted arrays
Advanced methods in molecular biology - Introduction to microarrays
Other common in situ synthesised
oligonucleotide arrays
• NimbleGen; similar to Affymetrix arrays but rather than using
photolithography masks to direct the photochemistry of oligo synthesis,
specialised aluminium digital mirrors devices automatically focus the
light onto the correct part of the array
• Inkjet microarrays; uses the same nozzles developed to spray ink
droplets in printers to fire appropriately sized droplets of A, C, G or T
nucleotides to the correct part of the microarray. Uses chemical means
to de-protect the preceeding base in the oligo to allow formation of a
new phoso-di-ester bond.
In situ oligonucleotide arrays are not used in ‘two colour competitive
hybridisations’ rather the experimental and reference samples are
labeled with the same fluorophore and each hybridised to its own micro
array
Advanced methods in molecular biology - Introduction to microarrays
Basic microarray experimental
summary for transcriptome
analysis
samples
RNA purification and
cDNA preparation
Fluorescent
labeling of
cDNA
Hybridisation
to microarray
and washes
Data collection and analysis
Advanced methods in molecular biology - Introduction to microarrays
Data Analysis (basic)
Microarray datasets are very large!!!!
Commercial microarrays come with analysis softwares and/or services
that contain algorithms that deal with such issues
• Data flagging; checking that each spot/ feature on the array is OK e.g.
obscured by dust or has poorly hybridised, and removing bad data
points.
• Data transformation; microarray data is most often given as fold
expression changes, that are best represented in Log2 space
• Background correction; a method to determine the quality of the
hybridisation either across a single microarray or between microarrays,
that take this into account when measuring fluoresence intensity of the
spot/ feature itself
Advanced methods in molecular biology - Introduction to microarrays
Data Analysis (basic)
• Normalisation; a method to ensure that the differences in gene
expression between samples are not artifactual e.g. hybridising twice as
much labeled sample in one case than the other.
- global normalisation/ scaling (assume that the median ratio (or
fold enrichment) in a data set = 1 or Log2 = 0) thereby allows calculation
of a ‘scaling factor’ to correct all the data points in one sample by that of
the other sample.
- local normalisation (each data point is normalised separately
according to predefined criteria such as fluorescence intensity)
e.g. LOWESS normalisation ( ‘ two colour ’ experiments) or
QUANTILE (for ‘single channel’ arrays).
Advanced methods in molecular biology - Introduction to microarrays
Data Analysis (basic)
LOWESS normalisation
Log2 expression fold
change
Raw data
After LOWESS
0
Fluorescence intensity
Note that the bais caused by low fluorescence
intensity has been corrected after LOWESS
normalisation
Advanced methods in molecular biology - Introduction to microarrays
Data Analysis (basic)
• Identifying trends in microarray data; e.g. hierarchical clustering and
visulalisation algorithms/ tools such as ‘ClusterTreeview;
Individual genes (spots/
feature on the microarray)
Replicate experiments (i.e
microarrays)
Advanced methods in molecular biology - Introduction to microarrays
MICROARRAY DATA STANDARDISTION
Microarray datasets are large and diverse (e.g. what type of array was
used, how many replicates) making the exchange of data between
researchers difficult.
Therefore journals now require that any published microarray data is
deposited into an internationally recognized database called MIAME,
that aims to standardise all the data and make it esily avaialble to the
whole of the research community
Advanced methods in molecular biology - Introduction to microarrays
MICROARRAY APPLICATIONS
• TRANSCRIPT PROFLING; discussed above and was the first application of
microarrays to identify changes in mRNA expression between two samples on a
large scale. Also adaptable to assay non-coding RNA expression differences e.g.
miRNAs
• ARRAY COMPARATIVE GENOME HYBRIDISATION (arrayCGH)*; a method
of assaying (whole) genome content between samples e.g. large genomic
deletions or amplifications (copy number changes) often associated with
cancer
• CHROMATIN IMMUNOPRECIPITATION (ChIP)*; a protocol to detect the
interaction of specific protein with defined regions of the genome e.g.
specific transcription factor or epigenetic chromatin/ histone modifications
• SINGLE NUCLEOTIDE POLYMORPHISM (SNP) & ALTERNATIVE SPLICE SITE
DETECTION; specially designed sequence spot/ features on the array (e.g.
different SNPs or known/predicted exon boundaries) are used to assay
samples for their presence
Advanced methods in molecular biology - Introduction to microarrays
CGH
Patients DNA
A cytological technique were two
genomes (e.g. from a cancer
patient and a population of
unaffected
individuals)
are
flourescently and differentially
labeled, then
competitively
hybridised (hence ‘CGH’) to a
spread
of
metaphase
chromsomes. The ratio of
intensity of the two fluorophores
along the chromosomes length
reported either deletions or
amplifications of that part of the
patients genome
EFFECTIVE BUT CRUDE WITH LOW
RESOLUTION
Reference DNA
CGH
Metaphase chromosomes
Patient deletion
Patient amplification
Advanced methods in molecular biology - Introduction to microarrays
Array CGH
Replace the metaphase chromosomes with microarrays containing spots/ features that
correspond to segments (however large or small one requires) of the genomic DNA
e.g. Array CGH of the DNA of a brain tumour patient
Much greater level of detail e.g. one can detect deletion/ amplification of individual
genes as well as whole chromosomes (possible when using exon arrays to detect even
intra-geneic copy number variations).
Advanced methods in molecular biology - Introduction to microarrays
Chromatin immunoprecipitation (ChIP)
A method to assay whether specific proteins are interacting with discreet
regions of the DNA genome in intact cell nuclei e.g. gene promoters or
chromsome telomeres or centromeres
Proteins could be . . . .
SPECIFIC TRANCRIPTION FACTORS
MODIFIED HISTONE/ CHROMATIN VARIANTS
ASSOCIATED WITH FUNCTION e.g. DNA
replication or transcription
Advanced methods in molecular biology - Introduction to microarrays
Chromatin immunoprecipitation (ChIP)
Cells
Cross-link with
Formaldehyde and
sonicate to fragment
Hybridise to
genomic array
Immunoprecipitate
DNA-protein-Ab
complexes
IgG to any protein
e.g. histone or TF
Reverse
protein-DNA
crosslinks
Fluorescently
Label DNA
Extract DNA
QPCR quantitation
ChIP:Chip. A high-throughput assay of protein:genome interactions
Advanced methods in molecular biology - Introduction to microarrays
e.g. ChIP:Chip for REST TF on
chromosmal tiling arrays
Utilised a microarray consisting of 44 tiled
chromosomal regions of the human genome (~1%
of the total). Each tile is one spot/ feature on the
microarray
REST
RE1
Probed with ChIP DNA using an antibody
against the transcription factor REST, from a
variety of human cells lines
Advanced methods in molecular biology - Introduction to microarrays
e.g. ChIP:Chip for REST TF
REST ChIP:Chip data annotated over in a genome browser window
Raw Data
Normalised Data
Bruce et al., Gen. Res. (2009)
Genes
One can identify which genes the REST TF is binding proximally to
and is therefore likely to regulate
Advanced methods in molecular biology - Introduction to microarrays
e.g. ChIP:Chip for REST TF
Functional analysis e.g. after RNAi for REST
HBA1/2 locus
Luciferase siRNA
(48h)
REST siRNA
(48h)
Luciferase siRNA
L1CAM locus
REST siRNA
Combining the ChIP:Chip with functional
analysis details that not all REST-genome
interactions are the same!
Bruce et al., Gen. Res. (2009)
Advanced methods in molecular biology - Introduction to microarrays
e.g. ChIP:Chip for REST TF
Performing
hierarchical
clustering on the functional
RNAi data reveals REST
interacts
with
slightly
different sequence motifs
that determine its affinity
for bining genomic DNA and
regulating the transcription
of its target genes
Bruce et al., Gen. Res. (2009)
Advanced methods in molecular biology - Introduction to microarrays
Microarrays revolutionized biological
sciences research
Number of articles describing the use of
‘expression’ microarrays
• Microarrays were at the
vanguard of the genomic era
• They are highly sensitive
and versatile
• As
an
established
technique they are optimized
and accessible (financially) to
many researchers
• They’ve been responsible
for many fundamental and
applied
research
acheivements
However microarrays are being superceded by more
advanced technologies termed ‘next generation
sequencing’
Advanced methods in molecular biology - Introduction to microarrays
Next generation sequencing
Any DNA (e.g.
ChIP DNA, cDNA,
gDNA etc.
DNA clusters then sequenced by conventional
reversible terminanting fluorescent based sequencing
Advanced methods in molecular biology - Introduction to microarrays
Next generation sequencing
• The number of identical sequences read
is directly proportional to the abundance
of the DNA in the original sample. Hence
the method is quantitative!
• CRUCIALLY - no prior knowledge of the
sequence is required to quantify the
abundance of the DNA in the sample not true for microarrays (i.e. manufacture
of the arrays relies on knowing the
sequence or at least having a clone)
e.g. ChIP-Seq
• Therefore good for investigating organisms for which no genomic sequence
data is available
• HOWEVER still quite a preliminary technique very expensive when
compared to microarrays, putting it out of reach of most researchers. ALSO
generates vast amounts of data, all of which may not be relevant to the
researcher.
Advanced methods in molecular biology - Introduction to microarrays
Hence it’s highly probable that
microarrays will remain with use for
the foreseeable future!
Introduction to Microarrays
Alexander W. Bruce.
[email protected]
Room B182 Faculty of Science