Bioinf301 Microarray2

Measuring Gene Expression Part 2: 

Measuring Gene Expression Part 2 David Wishart Bioinformatics 301 david.wishart@ualberta.ca

Measuring Gene Expression: 

Measuring Gene Expression Differential Display Serial Analysis of Gene Expression (SAGE) RT-PCR (real-time PCR) Northern/Southern Blotting DNA Microarrays or Gene Chips

Microarrays: 

Microarrays

DNA Microarrays: 

DNA Microarrays Principle is to analyze gene (mRNA) or protein expression through large scale non-radioactive Northern (RNA) or Southern (DNA) hybridization analysis Essentially high throughput Northern Blotting method that uses Cy3 and Cy5 fluorescence for detection Allows expressional analysis of up to 20,000 genes simultaneously

Four Types of Microarrays: 

Four Types of Microarrays Photolithographically prepared short oligo (20-25 bp) arrays (1 colour) Spotted glass slide cDNA (500-1000 bp) arrays (2 colour) Spotted nylon cDNA (500-1000 bp) arrays (1 colour/radioactive) Spotted glass slide oligo (30-70 bp) arrays (1 or 2 colour)

Principles of 2 Colour Microarrays: 

Principles of 2 Colour Microarrays

Microarray Definition of Probe and Target: 

Microarray Definition of Probe and Target There are two acceptable and completely opposite definitions. We will use: Target = the DNA that is spotted on the array Probe = the DNA that is labeled with the fluorescent probe

Microarray Scanning: 

Microarray Scanning

2-Colour Microarray Principles: 

2-Colour Microarray Principles overlay images and normalize Laser 1 Laser 2 Scan and detect with confocal laser system Image process and analyze Green channel Red channel

Typical 2-Colour Data: 

Typical 2-Colour Data

Microarrays & Spot Colour: 

Microarrays & Spot Colour

Principles of 1 Colour Microarrays: 

Principles of 1 Colour Microarrays RT-PCR

Microarrays & Spot Colour: 

Microarrays & Spot Colour

Two Colour vs. One Colour: 

Two Colour vs. One Colour Two-colour hybridization eliminates artifacts due to variation in: quantity of DNA spotted stringency of hybridization local concentration of label However, both samples *must* label with equivalent efficiency Information is lost for genes not expressed in the reference or control sample

Two Colour vs. One Colour: 

Two Colour vs. One Colour One-colour hybridization may have artifacts due to variation in: quantity of DNA spotted stringency of hybridization local concentration of label However good quality control (QC) means, fewer artifacts less manipulation, lower cost reduced loss of information (due to reference sample transcript content)

Specific Arrays of Interest: 

Specific Arrays of Interest Home-made Spotted Oligo Arrays Made using glass slides, Operon oligos and robotic spotting equipment Amersham CodeLink Microarrays Made using specially treated slides, QC’d oligos and robotic spotting equipment Affymetrix Gene Chips Made using photolithographically produced systems with multi-copy oligos

Array Images: 

Array Images 2 colour 1 colour

Array Images: 

Array Images 2 colour 1 colour Affymetrix Gene Chip

Home-made Spotted Arrays: 

Home-made Spotted Arrays

Spotted Microarrays: 

Spotted Microarrays Probes are >100m and are usually deposited on glass Probes can be: oligos (usually >40mers) PCR fragments from cDNA/EST or genomic templates Not reused; 2-colour hybridizations

Standard Spotted Array: 

Standard Spotted Array

Home-made Microarrays: 

Home-made Microarrays

Common Home-made Microarray Errors: 

Common Home-made Microarray Errors Irregular Spot Comet Tail Streaking Hi Background Low Intensity A Good Array

Testing Reproducibility: 

Testing Reproducibility Breast tumor tissue biopsy mRNA prepared using standard methods Control sample made from pooled mRNA from several cell types 3 RNA samples prepared from 1 tissue source – arrayed onto two sets of home-made chips from different suppliers Conducted pairwise comparison of intensity correlations & no. of spots

Home-made Arrays: 

Home-made Arrays 1) 2) 3) Oligo Microarray 1

Home-made Arrays: 

Home-made Arrays 1) 2) 3) Oligo Microarray 2

Advantages to Home-made Systems: 

Advantages to Home-made Systems Cheapest method to produce arrays ($100 to $300/slide) Allows lab full control over design and printing of arrays (customizable) Allows quick adaptation to new technologies, new probe sets Allows more control over analysis

Disadvantages to Home-made Systems: 

Disadvantages to Home-made Systems Quality and quality-control of oligo probe set is highly variable Quality of spotting and spot geometry is highly variable Technology is very advanced, difficult and expensive to maintain (robotics) Reproducibility is poor

Amersham CodeLink Arrays: 

Amersham CodeLink Arrays

Amersham CodeLink Arrays: 

Amersham CodeLink Arrays Amersham synthesizes its 30-nucleotide oligos offline, tests them by mass spectrometry, deposits them on specially coated array, and then assays them for quality control Uses a special Flex Chamber™—a disposable hybridization chamber already attached to the slide to improve hybridization consistency

Amersham CodeLinkOligo Chip: 

Amersham CodeLink Oligo Chip Glass Hydrophilic polymer DNA

CodeLink Special Coating: 

CodeLink Special Coating Most glass substrates are quite hydrophobic This hydrophobicity affects the local binding and surface chemistry of most glass-slide chips making most of the attached DNA oligo inaccessible Coating the slide with a hydrophilic polymer allows the cDNA to pair up with the substrate oligos much better

Amersham Microarrays: 

Amersham Microarrays RT-PCR

Slide34: 

Morphology Does Not Affect Dynamic Range CodeLink Bioarrays Can Achieve Linearity Across 3 Logs* *Data obtained from cRNA dilution series. The red line indicates the signal level for non-spiked target. Error bars represent one standard deviation for each mean (n=18) signal

Testing Reproducibility: 

Testing Reproducibility Breast tumor tissue biopsy mRNA prepared using standard methods 3 RNA samples prepared from 1 tissue source – arrayed onto 3 different sets of CodeLink chips Conducted pairwise comparison of intensity correlations, intensity ratio correlations & number of “passed” spots

Intensity, Pairwise Comparisons: 

Intensity, Pairwise Comparisons 1) 2) 3) Amersham Slides

Slide37: 

1) 2) 3) Ratio, Pairwise Comparisons Amersham Slides

General Comparison: 

General Comparison Amersham Intensity Amersham Ratio Vancouver Calgary I Calgary II

Comparative Accuracy: 

Comparative Accuracy GENES hENT1 hENT2 hCNT1 hCNT2 dck ER Expression Pattern TaqMan + + - - + + Expression Pattern Operon - - - + - - Expression Pattern Amersham + + - - + + RT-PCR Spotted Array CodeLink

CodeLink Advantages: 

CodeLink Advantages Exceptional reproducibility because of: careful probe design QC of oligo preparations and spotting high proportion of oligo binding to cDNA substrate due to hydrophilic coating well controlled/uniform hybridization Allows users to continue using same scanners/software as in spotted arrays

CodeLinkDisadvantages: 

CodeLink Disadvantages Lack of flexibility or customizability (users depend on Amersham to provide & design chips) Dependent on proprietary kits and reagents More expensive than spotted arrays ($700/chip)

Cost per Sample in Triplicate: 

Cost per Sample in Triplicate Amersham Slides (single channel) $2000 Vancouver Spotted Arrays (two colour) $800 Calgary Spotted Arrays (two colour) $1100

Affymetrix Gene Chips: 

Affymetrix Gene Chips Chips are 1.7 cm2 400,000 oligo probe pairs Probe “spots” are 20 x 20 Each probe is 25 bases long 11-20 “match” probes and 11-20 “mismatch” probes per gene

Affymetrix Gene Chip: 

Affymetrix Gene Chip

Affy Chip: 

Affy Chip A C T G C A C T G A . . A C T G C A C C G A . . C A G T A C C A C C . . C A G T A C C G C C . . G T A C C T T G T C . . G T A C C T T A T C . . A T C C A G G A A T . . A T C C A G G C A T . . T A T T A A A G C A . . T A T T A A A T C A . . T G A A T G A C A G . . T G A A T G A G A G . . Match probe 1 Mis-Match probe 1 Match probe 14 Mis-Match probe 14

Affy Chip: 

Affy Chip 11-20 probes for each gene/EST Each probe is 25 bases long 1 has exact match, the other is mismatched in the middle base Match (M) and mismatch (MM) pairs are placed next to each other Expression levels calculated using intensity difference between M & MM for all probe pairs

Affymetrix Hybridization: 

Affymetrix Hybridization

Affy Chips: 

Affy Chips

Affy Chips: 

Affy Chips match mismatch match mismatch

Affy Chips: 

Affy Chips

Comparison of Affymetrix and Spotted cDNA Arrays: 

Comparison of Affymetrix and Spotted cDNA Arrays 161 620 matched pairs of measurements from 56 cell lines Spotted Array Affymetrix Kuo et al. (2002) Bioinformatics

Affymetrix GeneChip Advantages: 

Affymetrix GeneChip Advantages High precision because of: careful probe design up to 20 probes per gene up to 20 mismatch probes Very precise measurements Very high density (500,000 elements/array)

Affymetrix GeneChipsDisadvantages: 

Affymetrix GeneChips Disadvantages Inflexible: each array requires custom photolithographic masks More expensive than spotted arrays ($1000-$1200 per chip) Proprietary technology not all algorithms, information public only one manufacturer of readers, etc.

General Comments: 

General Comments Spotted arrays are still wildly popular and widely used – a great learning tool for expression analysis Spotted arrays are generally unreliable and provide only gross indications of RNA expression Commercial systems (CodeLink and Affy) offer much greater reliability but are expensive & inflexible

Microarray Production: 

Microarray Production Probe design and selection Printing RNA extraction Labeling Hybridization and washing Scanning Data analysis

Probe Design & Selection: 

Probe Design & Selection Synthetic oligos 25-70 bases in length Choose sequences complementary to mRNA of interest Random base distribution and average GC content for organism Avoid long A+T or G+C rich regions Minimize internal secondary structure (hairpins or other loops) 1 M salt + 65 oC thermostability

Probe Design & Selection: 

Probe Design & Selection Design and select oligo sequences that are less than 75% identical to existing genes elsewhere in the genome (i.e. do a BLAST search) Sequences with >75% sequence identity to other sequences will cross-hybridize – leading to confounding results

Cross-hybridization: 

Cross-hybridization Xu et al. (2001) Gene hybridization intensity sequence similarity Analysis of a cross-hybridization within the CYP450 superfamily

Microarray Printing: 

Microarray Printing

Microarray Printing: 

Microarray Printing Probes are deposited by robots using: piezo-electric jets microcapillaries split or solid pins Coated glass is the most common substrate aminosilane, poly-lysine, etc. give non-covalent linkages covalent linkage is possible with modified oligos + aldehyde (etc.) coatings

RNA Extraction: 

RNA Extraction RNA is extremely unstable Probably the most problematic step in all microarray analysis RNA is extracted as “total RNA” only 1-2% is mRNA remainder is rRNA, tRNA, etc. RNA extracted from tissue is often very heterogeneous (many cells and cell types) – watch selectivity

Laser Capture Microdissection: 

Laser Capture Microdissection Cells of interest are visually selected and exposed to an IR laser, which adheres them to a transfer film arcturus.com

RNA Labeling: 

RNA Labeling Common source of systematic error (freshness, contaminants) Direct labeling fluorescent nucleotides are incorporated during reverse transcription (“first strand”) Indirect labeling reactive nucleotides (aminoallyl-dUTP) are incorporated during RT; first strand product is mixed with reactive fluorescent dyes that bind to amino group

Direct Labeling: 

Direct Labeling Cy3-ATP Cy5

Indirect Labeling: 

Indirect Labeling aminoallyl-dUTP

Hybridization: 

Hybridization Stringency of hybridization is affected by ions, detergents, formamide, temperature, time... Hybridization may be an important source of systematic error Automated hybridization systems exist; value is debatable

How Many Replicates?: 

How Many Replicates? Lee et al. (2000) PNAS Substantial error when only one array analyzed, standard is to use 3 replicates Singletons Duplicates 3X

What Types of Replicates?: 

What Types of Replicates? Biological replicates Technical replicates Biological replication is most important because it includes all of the potential sources for error

Microarray Production: 

Microarray Production Probe design and selection Printing RNA extraction Labeling Hybridization and washing Scanning Data analysis