1. Identification of Arabidopsis defense- and infection-related genes
DNA Microarray Analysis
Carine Denoux
Julia Dewdney

2. Steps for obtaining Microarray Data
Arabidopsis & pathogen experiment
Total RNA isolation & quality controls
Labeled cRNA target synthesis & quality controls
Probe array Hybridization
Probe array analysis 
Data analysis
6steps for obtaining the microarray data
experiment in greenhouse on Arabidopsis

3. Arabidopsis & pathogen Experiment
Each person does they experiment or treatment.
Experiments are repeated 3 times with control
Maximum 14 samples per experiment, if not I need to split experiment in half
the result is significative with 3 repetition for each experiment
the experiments are Realized at different time points,
Infected with different pathogen (: Botrytis cinerea, Erysiphe orontii, Fusarium oxysporum, Pseudomonas fluorescens)
Or treated with defense-related signaling compounds (: salicylic acid, jasmonic acid, ethylene)

4. plant tissue (& fungus)
Wash 3x
Elute
Add ethanol
One sample
plant tissue (& fungus)
Grind
&
lyse
100mg
Total RNA
Bind tRNA
Centrifuge through
QIAshredder
Total RNA isolation
RNeasy Qiagen kit Total RNA isolation from Plants and fungi
I can start my work with tissues and I extract RNA with RNeasy Qiagen kit
or I start to work straight (=directement) with total RNA
is important to combine 2 sample of 100 mg to have efficiently concentration

5. Total RNA quality controls
Check the ratio A260/280 in Tris between 1.9 and 2.1 have a good purity
Assess the RNA integrity on Bioanalyser with agilent technologies
I need to check the quality of the total RNA
First, I take OD check the purity , she is ok when the ratio A260/280 is between 1.9 and 2.1
(no contamination by the protein)
Second time, I assess the RNA integrity on bioanalysor:

6. The Agilent 2100 BioAnalyzer
This instrument uses lab-on-a-chip technology to perform automated quality control by capillary electrophoresis
It improves RNA analysis:
Rapid visualization of sample quality (12 samples in 30 min)
High sensitivity & small amount ( ng RNA)
Reduced use and waste of hazardous chemicals
The system software provides
access to real-time data.
* Calculate the ratio of ribosomal bands in total RNA samples and show the percentage of ribosomal impurities in mRNA samples.
I need to explain the processes: gel dye mix depot, priming station, decontamining electrodes, loading RNA nano marker,ladder, samples, vortex chips..

7. RNA 6000 Nano Assay Loading : 1-Loading gel-dye mix 2-pressurize
3-Loading RNA 6000 Nano Marker
4- Loading ladder
5- Loading samples
6-Vortexing

8. Total RNA profile Gel with total RNA Electropherogram total RNA 28S
2 profile: 1 gel and 1 electropherogram
we can see on their profile integrety of RNA (if the RNA is degraded or not)

9. Labeled cRNA target synthesis
Total RNA
First & second
strand
cDNA synthesis
Cleanup of
double-stranded
cDNA
In vitro
transcription (IVT)
cRNA synthesis
Cleanup of
biotin-labeled
cRNA
procedure need 3 to 4 days, OD
bioanalysis
fragmentation OD
bioanalysis

10. cRNA Quality control Before fragmentation Before Frgt After Frgt After
4000
2000
1000
500
200
marker
I control on bioanalysor the cRNAbefor and after fragmentation
RNA 6000 nano marker
the marker is use to aligne all the gel and compare the profiles
the simple before framentation is a smear
the simple after fgtation expected(attendu) between 35 and 200 bp
After
fragmentation

11. Probe array Hybridization
To get hybridization Cocktail
Hybridize 16 hours at 45° F the test probe array
Washing and Staining the array on fluidic station
Scanning the test chip
Analyze data
Hybridize the real probe array
for the real probe array , hybridize 16 hours a 45 ° F with rotation

12. Chips Back Front Septa Plastic cartridge
Probe array on glass substrate

13. Fluidic station microarray staining buffer
Automatic wash with the fluidics station
we can use 1 or 2 fluidic station (if no body else need it )

14. Fluidic protocol-Eukaryotic target
manually we lose long time because many repetitives cycles in protocol
and each wash and stain are very variable
the automatic way is better

15. Probe array analysis Watch no bubbles on chip before scanning
Remove bubbles (50% array have bubbles)
refill in fluidic station or remove by pipetting
Scanning each array 2 times
Check image & data

16. Image Data
this is the image we can see on computer when the array is scannned

17. Image control in center
Control standard gene
standard gene expression positive of different eukaryotic species
control probe set is a hybridization control
Control probe set

18. Image control on corners
Image data
with grid
Intensity data
I check on 4 corner if the signal and grid match well
With the mach the computer give us the intensity data
and we can use software for analyze the data
Signal and grid match on 4 corners

19. Initial Data Analysis with MAS 5
MicroArray Suite #5
Pivot table:
Bio controls : probe set and standard genes
are indicative of hybridization and array sensitivity
Analyses table:
RawQ  noise control, 1 is low noise
> 5 array is not good for data analyses
SF : Scaling Factor  reflect the intensity
lower and the same across all arrays is better
we can process to clustering
We use the intensity data in resolver data base for comparative analyses

20. Conclusion Time of my work is between 2 and 3 weeks
Each control step is important  high cost of each sample
the time of my work is 3 weeks if I isolate total RNA or 2 weeks
each control steps is important because the cost is high 3000$ one chips

21. Summary Experiment carried out: At+OGs, At+Bc, Simone exp
At+Eo, Mary exp
At+Bc, Joulia exp & Simone exp
Future experiment:
At+Eo Julia exp
At+ Ps SAS JianPing exp,
At+Fo Andrew exp

22. Thanks to Frederick Ausubel Julia Dewdney Lisa Racki CGR staff:
Jennifer Couget
Shufen Meng