MICROARRAYS D’EXPRESSIÓ

ESTUDI DEL FACTOR DE TRANSCRIPCIÓ ASH2

M. Corominas: mcorominas@ub.edu

Spotted array experiment

1. Prepare sample.

Test Reference

2. Label with fluorescent dyes.

3. Combine cDNAs.

4. Print microarray.

5. Hybridize to microarray.

6. Scan.

Spotted microarrays rely on delivery technologies to place biologic material (purified cDNA, oligonucleotides) onto allocated locations of the chip.

(competitive hybridization: Cy3 vs Cy5)

Drosophila melanogaster

Wolpert (2001)

ash2- member of a trithorax group

-Polycomb (PcG) : transcriptional repression-trithorax (trxG) : transcripcional activation

Belongs to multiprotein chromatin remodeling complexes

Transcriptional Regulator

Plates from the Berkeley Drosophila Gene Collectionwith 384 wells (clones) each: DGC1.0 and 2.0

Aprox. 12000 genes in total

Direct PCR from Bacterial Growth using vector-specificprimers

Analysis of PCR resultsby electrophoresis

Spotting onslide

1) From full length cDNA

TYPES OF MICROARRAYS

2) From 400 bp amplicons

a) correspond to approximately 75% of genes predicted in release 3.1 (gene specific primers kindly donated by Incyte Genomics and Brian Oliver, NIH).

b) based on a novel annotation of the fly genome. It contains 21376 gene- specific probes.

Performed and available from Eurogentec. Carried out in collaboration (ZMBH, Univ. of Heidelberg; DKFZ, MPI Molecular GeneticsComputational Molecular Biology, Germany)

TYPES OF MICROARRAYS

3) From oligonucleotides

a) INDAC project: International Drosophila Array Consortium

www.indac.net

70 mer oligonucleotides designed towards the 3’ endof the genes (based on the 3.1 release) with specificalgorithms and synthesized by Illumina.

b) Qiagen/Operon oligo set

70 mer oligonucleotides representing 13,664 genes designed from release 3.1

already available in the Plataforma de TranscriptòmicaServeis Científico-Tècnics UB- PCB

- RNA samples:- total RNA- polyA+ RNA- T7 polymerase amplified RNA

- labeling method (competitive hybridization):- direct- indirect

MIAME describes the Minimum Information About a Microarray Experiment that is needed to enable the

interpretation of the results of the experiment unambiguously and potentially to reproduce the

experiment.

http://www.mged.org/Workgroups/MIAME/miame.html

- positive and negative controls

Production of cDNA chips

17 plates from the Berkeley Drosophila Gene Collectionwith 384 wells (clones) each.

Aprox. 5000 genes in total

Direct PCR fromBacterial Growth

Analysis of PCR resultsby electrophoresis

Spotting onslide

Hybridization of Chips

mutant flies (ash2) wild-type flies

Two-Step FluorescentLabelling

Trizol RNA Extraction& Poly A+ Purification

mRNA mRNA

Cy5 test sample Cy3 control sample

Hybridize Slide

GenePix

Scanning of Chips

fluorescent intensities for each cDNA, spot or gene

Scan Slide 532 nm

fluorescent intensities for each cDNA, spot or gene

635nm

-Integrate Data-Filter Data-Adjust dye bias

-Calculate Ratios-Adjust Data-Set Thresholds

“Bad” Spots Filtering

- Is the process in which spots that don’t look right are discarded according to different criteria

GenePix discards data according to internal filters like: x % pixels > Median Background intensity

Convert Data 3.33 to further filter data. Spots were flagged as OK if:medianFx > mBx +/- XSD

- Spots must pass filtering for both channels

Distribution for Good spots at both wavelengths

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Adjusting Ratios

- Different measures for the ratios:- Ratio of Medians- Ratio of Means- Regression Ratio

-Log (base 2) the ratios :•Makes variation of intensities and ratios of intensities more  independent of absolute magnitude. •Gives a more realistic sense of variation.

- A Ratio measures how much sample cDNA over control cDNA we have of a given gene. This is: Ratio = Intensity sample / Intensity control

Multiple Experiment Comparison

- ratio (eg. Ratio of Medians)

- bad spots filtering methods

- adjust ratios:- mean centering- Normalization - main class centering

Modify data the same way in all experiments:

- We expect:- few genes upregulated- few genes downregulated- most genes unchanged (log2 Ratio = 0)

-Therefore:- a Normal distribution- with mean (all log2 Ratio ) = 0

-Draw distribution of Ratios and check mean:- if really not N: filter bad spots better

try to Normalize (mean = 0; SD = 1)discard experiment

- if close to N: adjust mean (product or sum)Normalize (0; 1)

Norm log Ratio of Medians

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Multiple Experiment Comparison

Set method to select up or downregulated genes

- higly subjective method like fold-change (eg. two, three)

- semi-statistical method like Mean ± xSD

- statistical method like SAM:- missing values imputed using a K-nearest Neighbor- computes a statistic- set threshold for statistic (to call significant genes)- will give you a FDR- set fold-change threshold

Results

4163 different genes with FBgn

(SAM INPUT)

SAM 2.5% FDR1.75 Foldchange

140 95

5139 different genes with FBgn in total

Filtering of BadSpots

Mean Corr. Coef0.88

- If a gene remains unchanged in the mutant (ash2I1):Ratio = F sample / F control = 1log2 Ratio = 0, because log2 1=0ash2 is not regulating this gene

-If a gene is downregulated in the mutant (ash2I1):Ratio = F sample / F control <1log2 Ratio <0, because log2 1=0ash2 is in activation pathway

-If a gene is upregulated in the mutant (ash2I1):Ratio = F sample / F control > 1log2 Ratio > 0, because log2 1=0ash2 is in repression pathway

Controls and Quality assesment

- Sequencing of some clones from the Collection plates

- RT-PCR of some genes in a semiquantitative way

- Western Blot

- Northern Blot

- Clonal Analysis

- in situ hybridization

- inmunolocalization

RT-PCR+ = wt - = ash2

ASH2

Classification according to GO (Gene Ontology)

- Gene Ontology is a “controlled vocabulary that can be applied to all eukaryotes “. Each gene product is classi- fied in one or more categories.

- Is distribution of missexpressed genes significantly different from the one of our initial set of genes?

- maybe ash2 acts predominantly upon a group of genes of similar function or pathway

18S28S 3

Fluorescence

Time (seconds)

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19 24 29 34 39 44 49 54 59 64 69

Operon D. melanogaster Array

- 10 A. thaliana oligos (TIGR spikes) - each printed 4 times by pin = 640 spots- 12 D. melanogaster oligos - each printed 17 times = 204 spots

16416 spots

- 12 Randomly Generated Negative Controls – printed several times = 188 spots- 352 Empty spots- 449 Buffer spots

14593 70mer probes representing 13664 genes and 17899 transcripts

POSITIVE CONTROLS

NEGATIVE CONTROLS

(hybridized with aRNA ISOash2I1 vs ISO)

ANALYSIS LAYOUT

2 TIFF images (Cy3 & Cy5) GAL file (gene matrix)

1 GPR file for experiment

Output

TIGR Express Converter 1.4.1

1 MEV file for experiment

Output

Input

GenePix Pro 4.0 Image analysis

Input

1 MEV file for experiment (total=5)

TIGR MIDAS- Each experiment analyzed independently- Background filter applied- Normalization applied: Lowess (LOC) for each experiment independently

Input

Input

EXCEL & TIGR MEV

- Spike-in, negative and positive control Check- MA Plots- Experiment Comparison (Scatter Plots)- Relevant Genes Finding

TIGR spike-in Mix

On chip: 10 A. thaliana oligos spotted 64 times each (4 times by pin)

To add to labeling reaction: In vitro synthesized RNA from eachgene at different proportions and quantities:

GENE Ratio Mix A Mix BRCA 1 to 1 5000 5000Cab 1 to 1 2000 2000RbcL 1 to 1 500 5000Ltp4 1 to 1 20 20Ltp6 2 to 1 3000 1500PRK 2 to 1 500 250TIM 2 to 1 100 50Nac 1 to 3 10 30RCP 1 to 3 200 600XCP 1 to 3 1000 3000

pg in 2 ul of:

We can use the spikes to assess quality of experiment and analysis

For Amplification experimentswe use the spikes diluted 1:500

TIGR spikes MA plot from an experiment with total RNA

DOO-016TIGR Spikes MA Plot

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log2(Cy5/Cy3)

log2(Cy5*Cy3)

RCA (11)

CAB (11)

rbcL (11)

LTP4 (11)

XCP2 (13)

RCP1 (13)

NAC1 (13)

Ltp6 (21)

PRKase (21)

TIM (21)

3 to 1 ratio

1 to 2 ratio

Experimental procedure and analysis seems good(spikes fall where expected)

Operon Arrays InsetsISO ash2I1 vs ISO

L3 total RNA aRNA from L3 total RNA

- 60ug indirectly labelled - 2 ug amplified to 70ug in 4h- 20ug of labelled aRNA

TIGR Spikes

Amplification Test:totalRNA vs aRNA log2ratios

Correlation coef = 0.94

Biological Replicates

Fluorescence

Time (seconds)

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19 24 29 34 39 44 49 54 59 64 69

18S28S

Fluorescence

Time (seconds)

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19 24 29 34 39 44 49 54 59 64 69

Fluorescence

Time (seconds)

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19 24 29 34 39 44 49 54 59 64 69

18S28S

Fluorescence

Time (seconds)

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19 24 29 34 39 44 49 54 59 64 69

REPLICATE 1 REPLICATE 2

Biological ReplicatesMicroarray Insets

REPLICATE 1 REPLICATE 2

Amplified TIGR spikes(diluted 1:100) together with probes

Biological ReplicatesReplicate 1 vs Rplicate 2 log2ratios

Correlation coef = 0.92