Jan 18, 2016
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
0
20
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60
80
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120
5
5.8
6.6
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.6
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.2 13
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.8
log (F Median - B)
Nu
mb
er
in e
ach
cla
ss
log(F
63
5M
edia
n-B
63
5)
log(F
53
2M
edia
n-B
53
2)
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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-7
-5.9
-4.8
-3.7
-2.6
-1.5
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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
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)
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
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
-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)
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)
0.0
2.5
5.0
7.5
10.0
12.5
19 24 29 34 39 44 49 54 59 64 69
18S28S
Fluorescence
Time (seconds)
0
5
10
15
20
25
30
35
19 24 29 34 39 44 49 54 59 64 69
Fluorescence
Time (seconds)
0.0
2.5
5.0
7.5
10.0
12.5
19 24 29 34 39 44 49 54 59 64 69
18S28S
Fluorescence
Time (seconds)
0
5
10
15
20
25
30
35
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