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 6steps for obtaining the microarray data experiment in greenhouse on Arabidopsis

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)

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

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:

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. * 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..

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 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)

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

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

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

Chips Back Front Septa Plastic cartridge Probe array on glass substrate

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 )

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

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 this is the image we can see on computer when the array is scannned

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

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

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

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

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