Transcript PowerPoint Presentation - Identification Arabidopsis

Identification of Arabidopsis
defense- and infection-related genes
DNA Microarray Analysis
Carine Denoux
Julia Dewdney
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
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
One sample
plant tissue (& fungus)
100mg
Total RNA isolation
Grind
&
lyse
100mg
Centrifuge through
QIAshredder
RNeasy Qiagen kit
Add ethanol
Bind tRNA
Total RNA isolation from
Plants and fungi
Total RNA
Wash 3x
Elute
Total RNA
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
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
(100-200ng RNA)
 Reduced use and waste of hazardous
chemicals
 The system software provides
access to real-time data.
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
Total RNA profile
Gel with total RNA
28S
18S
Electropherogram total RNA
Labeled cRNA target synthesis
Total RNA
First & second
strand
cDNA synthesis
In vitro
transcription (IVT)
cRNA synthesis
OD
bioanalysis
fragmentation
Cleanup of
double-stranded
cDNA
Cleanup of
biotin-labeled
cRNA
OD
bioanalysis
cRNA Quality control
Before fragmentation
Before Frgt
After Frgt
4000
2000
1000
500
200
marker
After
fragmentation
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
Chips
Plastic cartridge
Septa
Probe array on
glass substrate
Back
Front
Fluidic station
microarray
staining buffer
Fluidic protocol-Eukaryotic target
Standard Format EukGE-WS2
Post Hyb : Wash #1
10 cycles of 2 mixes/cycle with Wash Buffer A at 25¼C
Post Hyb : Wash #2
4 cycles of 15 mixes/cycle with Wash Buffer B at 50¼C
Stain
Stain the probe array for 10 minutes in Streptavidin Solution.at
25¼C
Post stain : Wash
10 cycles of 4 mixes/cycle with Wash Buffer A at 30¼C
2 nd stain
Stain the probe array for 10 minutes in antibody solution.at 25¼C
3 rd stain
Stain the probe array for 10 minutes in Streptavidin Solution at
25¼C
Final Wash
15 cycles of 4 mixes/cycle with Wash Buffer A at 30¼C. The
holding temperature is 25¼C
tandard Format
¥ Wash Buffer A = non-stringent wash buffer
¥ Wash Buffer B = stringent wash buffer
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
Image Data
Image control in center
Control standard gene
Control probe set
Image control on corners
Image data
with grid
Signal and grid match on 4 corners
Intensity data
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 use the intensity data in resolver data base for comparative analyses
Conclusion
• Time of my work is between 2 and 3 weeks
• Each control step is important  high cost of
each sample
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
Thanks to
Frederick Ausubel
Julia Dewdney
Lisa Racki
CGR staff:
Jennifer Couget
Shufen Meng