CS177 Lecture 11 Experimental Methods (PCR, X-ray crystallography, Microarrays)
Introduction to Microarrays Alexander W. Bruce. [email protected] Lecture theatre C3, Faculty of...
-
Upload
leonard-bryant -
Category
Documents
-
view
219 -
download
0
Transcript of Introduction to Microarrays Alexander W. Bruce. [email protected] Lecture theatre C3, Faculty of...
Introduction to Microarrays
Alexander W. [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
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 . . . . .
Advanced methods in molecular biology - Introduction to microarrays
Not necessarily in that order . . .
Central dogmaAdvanced methods in molecular biology - Introduction to microarrays
GENOME
TRANSCRIPTOME
PROTEOME
DNA/ genes
RNA/ transcripts
protein
TRANSCRIPTOME
How can we measure components of the TRANSCRIPTOME i.e. transcripts?
Nucleic acid hybridisation
Advanced methods in molecular biology - Introduction to microarrays
Duplex of complementary base-
pairing 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
How can multiple specific nucleic acids (e.g. mRNAs) be detected in a sample at the
same time ?
Advanced methods in molecular biology - Introduction to microarrays
Answer: invert the system i.e. immobilise the probes and label the sample
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
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
Advanced methods in molecular biology - Introduction to microarrays
The ancestral microarray !?
Method improved and expanded to include 4000 cDNA bacterial clones of human cDNAs automatically spotted as liquid cultures onto membranes before lysis.
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
Advanced methods in molecular biology - Introduction to microarrays
Some commercial exploitation of this semi-quantitative technique e.g. ‘Clonetech expression blots’
Arraying PCR productsAdvanced methods in molecular biology - Introduction to microarrays
PCR amplifying cDNA from plasmid cDNA library collections
Cloning into a
common plasmid vector
cDNAs synthesised from mRNA
cDNA library
Organised cDNA library
Transform bacteria
PCR amplify specific cDNAs from each well with the same
primer pairs and spot on microarray
Single colony innoculation into multiwell plates
Patrick Brown
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]
Advanced methods in molecular biology - Introduction to microarrays
The first ‘true’ microarray
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’
COMPETITIVE HYBRIDISATIONS
Advanced methods in molecular biology - Introduction to microarrays
Experimental & Reference samples
Purified RNA
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.
cDNA synthesis incorporating different fluorescently labeled dNTPs for
experimental and reference RNAs
e.g.Cy3 or Cy5 dyes
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
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.SourceDepartment 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
Advanced methods in molecular biology - Introduction to microarrays
GENOME-WIDE MICROARRAY
Interrogation of the ENTIRE TRANSCRIPTOME in a single experiments
Refinements of microarray based techniques
Advanced methods in molecular biology - Introduction to microarrays
Sample cDNA fluorescent labeling methods
Cy3/ Cy5 coupled dNTPs
Poor rates of incorporation by reverse transcriptase enzymes due to ‘bulky’ Cy dye
moiety
Amino-allyl modified dNTPs are more efficiently incorporated into cDNA . . .
. . . and the fluorescent Cy dyes can then be coupled to this reactive amine after completion of
cDNA synthesis
Cy3/5
Refinements of microarray based techniques
Advanced methods in molecular biology - Introduction to microarrays
Array manufacture • traditionally double stranded (ds) 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 AA
Probe cDNAs generated by PCR where one of the oligo primers used has a 5’
amino modification
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
Spotted oligonucleotide arrays
Advanced methods in molecular biology - Introduction to microarrays
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
Spotted oligonucleotide microarraysAdvanced methods in molecular biology - Introduction to 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!
In situ synthesised oligonucleotide arrays
Advanced methods in molecular biology - Introduction to microarrays
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
Other common in situ synthesised oligonucleotide arrays
Advanced methods in molecular biology - Introduction to microarrays
• 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
Basic microarray experimental summary for transcriptome
analysis
Advanced methods in molecular biology - Introduction to microarrays
samplesRNA purification and
cDNA preparation
Fluorescent labeling of
cDNA
Hybridisation to
microarray and washes
Data collection and analysis
Data Analysis (basic)
Advanced methods in molecular biology - Introduction to microarrays
Microarray datasets are very large!!!!
• Data transformation; microarray data is most often given as fold expression changes, that are best represented in Log2 space
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.
• 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
Data Analysis (basic)
Advanced methods in molecular biology - Introduction to microarrays
• 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).
Data Analysis (basic)
Advanced methods in molecular biology - Introduction to microarrays
LOWESS normalisation
Raw data After LOWESS
Fluorescence intensity
0
Log2
exp
ress
ion
fold
ch
ange
Note that the bais caused by low fluorescence intensity has been corrected after LOWESS
normalisation
Data Analysis (basic)
Advanced methods in molecular biology - Introduction to microarrays
• 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)
MICROARRAY DATA STANDARDISTIONAdvanced methods in molecular biology - Introduction to microarrays
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
MICROARRAY APPLICATIONSAdvanced methods in molecular biology - Introduction to microarrays
• 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
CGHAdvanced methods in molecular biology - Introduction to microarrays
EFFECTIVE BUT CRUDE WITH LOW RESOLUTION
Metaphase chromosomes
Patients DNA Reference DNA
CGH
Patient deletion
Patient amplification
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
Advanced methods in molecular biology - Introduction to microarrays
Array CGHReplace the metaphase chromosomes with microarrays containing spots/ features that correspond to segments (however large or small one requires) of the genomic DNA
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).
e.g. Array CGH of the DNA of a brain tumour patient
Chromatin immunoprecipitation (ChIP)Advanced methods in molecular biology - Introduction to microarrays
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
Chromatin immunoprecipitation (ChIP)Advanced methods in molecular biology - Introduction to microarrays
Cells
Extract DNA
QPCR quantitation
Cross-link with Formaldehyde and sonicate to
fragment
Reverse protein-DNA crosslinks
FluorescentlyLabel DNA
Hybridise to genomic array
ChIP:Chip. A high-throughput assay of protein:genome interactions
Immunoprecipitate DNA-protein-Ab
complexes
IgG to any proteine.g. histone or TF
e.g. ChIP:Chip for REST TF on chromosmal tiling arrays
Advanced methods in molecular biology - Introduction to microarrays
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
e.g. ChIP:Chip for REST TFAdvanced methods in molecular biology - Introduction to microarrays
REST ChIP:Chip data annotated over in a genome browser window
Raw Data
Normalised Data
Genes
One can identify which genes the REST TF is binding proximally to and is therefore likely to regulate
Bruce et al., Gen. Res. (2009)
e.g. ChIP:Chip for REST TFAdvanced methods in molecular biology - Introduction to microarrays
Functional analysis e.g. after RNAi for RESTHBA1/2 locus Luciferase
siRNA(48h)
REST siRNA(48h)
L1CAM locus Luciferase siRNA
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)
e.g. ChIP:Chip for REST TFAdvanced methods in molecular biology - Introduction to microarrays
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)
Number of articles describing the use of ‘expression’ microarrays
Microarrays revolutionized biological sciences research
Advanced methods in molecular biology - Introduction to 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’
Next generation sequencingAdvanced methods in molecular biology - Introduction to microarrays
Any DNA (e.g. ChIP DNA, cDNA, gDNA etc.
DNA clusters then sequenced by conventional reversible terminanting fluorescent based sequencing
Next generation sequencingAdvanced methods in molecular biology - Introduction to microarrays
• The number of identical sequences read is directly proportional to the abundance of the DNA in the original sample. Hence the method is quantitative!
e.g. ChIP-Seq
• 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)• 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.
Hence it’s highly probable that microarrays will remain with use for
the foreseeable future!
Advanced methods in molecular biology - Introduction to microarrays