PowerPoint Presentation - Identification Arabidopsis defencse-related genes

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

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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

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Total RNA isolation
RNeasy Qiagen kitTotal RNA isolation from
Plants and fungi
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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

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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.

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RNA 6000 Nano Assay
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Total RNA profile
Electropherogram total RNA
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Labeled cRNA target synthesis
First second strand cDNA synthesis
Total RNA
Cleanup of double-stranded cDNA

In vitro transcription (IVT) cRNA synthesis
Cleanup of biotin-labeled cRNA
OD bioanalysis
fragmentation
OD bioanalysis
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cRNA Quality control
Before fragmentation
Before Frgt After Frgt
4000 2000 1000 500 200 marker
After fragmentation
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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

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Chips
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Fluidic station
microarray
staining buffer
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Fluidic protocol-Eukaryotic target
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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

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Image Data
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Image control in center
Control standard gene
Control probe set
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Image control on corners
Image data
with grid
Signal and grid match on 4 corners
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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
• gt 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
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Conclusion

• Time of my work is between 2 and 3 weeks
• Each control step is important ? high cost of
  each sample

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Summary

• Experiment carried out
• AtOGs, AtBc, Simone exp
• AtEo, Mary exp
• AtBc, Joulia exp Simone exp
• Future experiment
• AtEo Julia exp
• At Ps SAS JianPing exp,
• AtFo Andrew exp

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Thanks to

• Frederick Ausubel
• Julia Dewdney
• Lisa Racki
• CGR staff
• Jennifer Couget
• Shufen Meng