MICROARRAYS D’EXPRESSIÓ

ESTUDI DE REGULADORS DE LA TRANSCRIPCIÓ DE LA FAMILIA trxG

M. Corominas: mcorominas@ub.edu

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

(competitive hybridization: Cy3 vs Cy5)

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

- 90% amplification- Single product in most PCRs

10,000 3,0001,000

10,000 3,0001,000

1,000

10,000 3,000

10,000 3,000

1,000

21,226 5,148 2,000

21,226 5,148 2,000

21,226 5,148 2,000

21,226 5,148 2,000

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

18S28S 3

Fluorescence

Time (seconds)

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

19 24 29 34 39 44 49 54 59 64 69

Hybridization of Chips

mutant flies (ash2) wild-type flies

FluorescentLabelling

RNA Extraction

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

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)

Amplification Test:totalRNA vs aRNA log2ratios

Correlation coef = 0.94

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

-3

-2

-1

0

1

2

3

27 32 37 42 47

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)

“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

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

- 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

0

1

2

3

4

5

6

7

-7

-5.9

-4.8

-3.7

-2.6

-1.5

-0.4

0.7

1.8

2.9 4

5.1

6.2

log Ratio of Medians Class

% G

en

es in

Cla

ss

Experiment 1 Experiment 3Experiment 2 Experiment 4

Multiple Experiment Comparison

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

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

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 trxG genes act predominantly upon a group of genes of similar function or pathway?

CRG-UPF-IMIM

Roderic GuigóEnrique Blanco

Departament de Genètica:

Florenci Serras

Montserrat Corominas

Isabel Almudí

Mireia Angulo

Sergi Beltran

Cristina Pallarès

Miguel Pignatelli

Adrià Punset

Marta Sesé

Plataforma de TranscriptòmicaParc Científic- SCT-UB

Lídia Sevilla