Affymetrix GeneChip Soybean Genome Array

Distinct biphasic mRNA changes in response to Asian soybean rust infection.

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Expression profiling in Rpp2-resistant (PI230970) and susceptible (Embrapa-48) plant lines to soybean rust from infection to symptom development

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Transcription profiling of Rpp2-resistant (PI230970) and susceptible (Embrapa-48) soybean cultivars to soybean rust from infection to symptom development

Distinct Biphasic mRNA Changes in Response to Asian Soybean Rust Infection

Expression profiling in Rpp2-resistant (PI230970) and susceptible (Embrapa-48) plant lines to soybean rust from infection to symptom development


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-Unknown

P-TABM-1247

Leaf samples were  immediately frozen in liquid nitrogen and stored at -80 °C. Upon further processing, leaf tissue was ground in liquid nitrogen and RNA was extracted using 1mL RNAwiz (Ambion, Austin, TX) according to manufacturer’s protocol. After precipitation of RNA in 2-propanol, the samples were stored at -80 °C and then shipped to Iowa State University where the extraction protocol was completed and the RNA samples were resuspended in 100 ?L DEPC-treated distilled water containing 2 U/?L RNAseOUT (Invitrogen, Carlsbad, CA). RNA samples were precipitated overnight in 2M lithium chloride (final concentration) at -20 °C followed by a 30 min centrifugation at 7600 x g at 4°C and an ethanol wash after which the RNA pellet was resuspended in 100 ?L DEPC-treated distilled water containing 2 U/?L RNAseOUT. The RNA samples were further purified using RNeasy purification (Qiagen, Valencia, CA) according to the manufacturer’s protocol and eluted in 30 ?L DEPC-treated water.

P-TABM-1248

Soybean plants at V6-8 growth stage, PI230970 (Rpp2, resistant) and Embrapa-48 (susceptible)

P-TABM-1250

A microarray experiment was designed to assess soybean gene expression during critical stages of Soybean Rust infection in resistant (PI230970, Rpp2) and susceptible (Embrapa-48) plants. A Brazilian greenhouse-maintained field isolate of Soybean Rust was used. In each interaction, gene expression was assayed in leaves inoculated with a spore suspension of SBR containing 0.5% Tween-20 and in mock-inoculated leaves sprayed with the Tween-20 solution. Two trifoliolate leaves were harvested from plants at the V6-8 growth stage at 6, 12, 18, 24, 36, 48, 72, 96, 120 and 168 hours after inoculation for RNA extraction and assessed for soybean mRNA transcript abundance. The experiment was replicated three times and samples were randomly processed by replication.

P-TABM-1251

Prior to performing the analysis, the MAS5.0 data was transformed by taking the log, base 2, of every observation. Each observation then had the median of all the observations from that slide subtracted from it. Thus, each chip was median centered such that the median of all the observations from each slide was now zero. Mixed model analysis was then performed separately for each gene modeling all three treatments and their interactions as fixed classification variables in SAS (SAS Institute, Cary, NC). Data analysis for both cultivars was performed together to pool the variance and increase the denominator degrees of freedom. This is possible since separate analysis of both cultivars did not significantly change the results. However, statistical significance was inferred by comparing an SBR infected sample at a given time point to the mock treated sample at that same time point within each cultivar. F tests, resulting in p-values, were performed to test for treatment effects. A q-value adjustment was then computed for each p-value using the method described by Storey and Tibshirani (PNAS (2003) 100, 9440-9445).

Transcription profiling of Rpp2-resistant (PI230970) and susceptible (Embrapa-48) soybean cultivars to soybean rust from infection to symptom development

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