Transcript Spotted

MICROARRAYS
D’EXPRESSIÓ
ESTUDI DE REGULADORS DE LA
TRANSCRIPCIÓ DE LA FAMILIA trxG
M. Corominas: [email protected]
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
- 90% amplification
- Single product in most PCRs
21,226
5,148
2,000
17 plates from the Berkeley Drosophila Gene Collection
with 384 wells (clones) each.
21,226
5,148
2,000
10,0
00
3,00
1,000
0
10,0
00
1,000
3,00
0
21,226
5,148
2,000
10,00
0
21,226
5,148
2,000
10,0
00
Aprox. 5000 genes in total
Direct PCR from
Bacterial Growth
Analysis of PCR results
by electrophoresis
1,000
3,000
1,000
3,00
0
Spotting on
slide
Operon D. melanogaster Array
16416 spots
14593 70mer probes representing 13664 genes and 17899 transcripts
POSITIVE CONTROLS
- 10 A. thaliana oligos (TIGR spikes) - each printed 4 times by pin = 640 spots
- 12 D. melanogaster oligos - each printed 17 times = 204 spots
NEGATIVE CONTROLS
- 12 Randomly Generated Negative Controls – printed several times = 188 spots
- 352 Empty spots
- 449 Buffer spots
20.0
17.5
15.0
12.5
7.5
5.0
2.5
0.0
19
24
29
34
39
3
18S
28S
Fluorescence
10.0
44
Time (seconds)
49
54
59
64
69
Hybridization of Chips
mutant flies (ash2)
wild-type flies
RNA Extraction
mRNA
mRNA
Fluorescent
Labelling
Cy5 test sample
Cy3 control sample
Hybridize Slide
Scanning of Chips
532 nm
Scan Slide
fluorescent intensities for GenePix
each cDNA, spot or gene
-Integrate Data
-Filter Data
-Adjust dye bias
635nm
fluorescent intensities for
each cDNA, spot or gene
-Calculate Ratios
-Adjust Data
-Set Thresholds
Operon D. melanogaster Array
16416 spots
14593 70mer probes representing 13664 genes and 17899 transcripts
POSITIVE CONTROLS
- 10 A. thaliana oligos (TIGR spikes) - each printed 4 times by pin = 640 spots
- 12 D. melanogaster oligos - each printed 17 times = 204 spots
NEGATIVE CONTROLS
- 12 Randomly Generated Negative Controls – printed several times = 188 spots
- 352 Empty spots
- 449 Buffer spots
(hybridized with aRNA ISOash2I1 vs ISO)
Amplification Test:
totalRNA vs aRNA log2ratios
Correlation coef = 0.94
TIGR spike-in Mix
We can use the spikes to assess quality of experiment and analysis
On chip: 10 A. thaliana oligos spotted 64 times each (4 times by pin)
To add to labeling reaction: In vitro synthesized RNA from each
gene at different proportions and quantities:
GENE
RCA
Cab
RbcL
Ltp4
Ltp6
PRK
TIM
Nac
RCP
XCP
Ratio
1 to 1
1 to 1
1 to 1
1 to 1
2 to 1
2 to 1
2 to 1
1 to 3
1 to 3
1 to 3
pg in 2 ul of:
Mix A
Mix B
5000
5000
2000
2000
500
5000
20
20
3000
1500
500
250
100
50
10
30
200
600
1000
3000
For Amplification experiments
we use the spikes diluted 1:500
TIGR spikes MA plot from an
experiment with total RNA
Experimental procedure and analysis seems good
(spikes fall where expected)
DOO-016TIGR Spikes MA Plot
3
2
RCA (11)
CAB (11)
1
rbcL (11)
log2(Cy5*Cy3)
LTP4 (11)
XCP2 (13)
RCP1 (13)
0
NAC1 (13)
Ltp6 (21)
PRKase (21)
-1
TIM (21)
3 to 1 ratio
1 to 2 ratio
-2
-3
27
32
37
log2(Cy5/Cy3)
42
47
“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
- A Ratio measures how much sample cDNA over control
cDNA we have of a given gene. This is:
Ratio = Intensity sample / Intensity control
- 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.
- 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)
Multiple Experiment Comparison
Norm log Ratio of Medians
Experiment 3
Experiment 4
7
6
5
4
3
2
log Ratio of Medians Class
6.2
5.1
4
2.9
1.8
0.7
-0.4
-1.5
-2.6
-3.7
-4.8
0
-5.9
1
-7
% Genes in Class
Experiment 1
Experiment 2
ANALYSIS LAYOUT
2 TIFF images (Cy3 & Cy5)
GAL file (gene matrix)
Input
GenePix Pro 4.0 Image analysis
Output
1 GPR file for experiment
Input
TIGR Express Converter 1.4.1
Output
1 MEV file for experiment
1 MEV file for experiment (total=5)
Input
TIGR MIDAS
- Each experiment analyzed independently
- Background filter applied
- Normalization applied: Lowess (LOC) for each experiment
independently
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
- in situ hybridization
- Northern Blot
- inmunolocalization
- Clonal Analysis
Classification according
to GO (Gene Ontology)
- Gene Ontology is a “controlled vocabulary that can be
applied to all eukaryotes “. Each gene product is classified 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
Departament de Genètica:
Florenci Serras
Roderic Guigó
Enrique Blanco
Montserrat Corominas
Isabel Almudí
Mireia Angulo
Sergi Beltran
Cristina Pallarès
Plataforma de Transcriptòmica
Parc Científic- SCT-UB
Miguel Pignatelli
Lídia Sevilla
Adrià Punset
Marta Sesé