|Application:||Gene expression microarray analysis|
|Number of samples:||28|
|Release date:||Feb 1 2009|
|Last update date:||Nov 22 2016|
|Dataset link||Transcriptional profiling of root-knot nematode induced feeding sites in cowpea (Vigna unguiculata L. Walp)|
Seeds of CB46 (resistant) and null-Rk (susceptible) cowpea genotypes were surface sterilized using 10% (v/v) bleach solution and were planted in growth pouches. Plants were grown under controlled environmental conditions of 26.7°C ± 0.5°C constant temperature and daily light/dark cycles of 16/8 hours. This temperature was used because it lies within the optimum temperature range of 26 – 28 °C for development and reproduction of M. incognita on cowpea in growth pouches. Each pouch was inoculated with 3000 J2 in 5 ml of deionized water, 12 days after planting (dap). Equal number of pouches was mock inoculated with 5 ml of deionized water to use as control. Nematode infected root tissue was excised using a sterile scalpel at 3 days post inoculation (dpi) and 9 dpi respectively under a magnifying glass and flash frozen immediately in liquid nitrogen. Approximately equal amount of root tissue was also excised and flash frozen from the control plants. The harvested tissue was stored in -80°C until RNA isolation. For 9 dpi samples 3 biological replicates were used for each treatment. So total number of soybean GeneChips used for 9dpi samples was 12. For 3 dpi samples two biological replicates were used for each treatment and a total of 8 GeneChips were used. Expression signals were first analyzed in Microarray Suite 5.0 software (MAS 5, Affymetrix Inc.) to determine the “present” probe set list. The detection algorithm uses probe pair intensities to generate a detection p-value and assign a “present”, “marginal”, or “absent” call. Each probe pair in a probe set has a potential vote in determining whether the measured transcript is or is not “present”. The vote is described by the discrimination score (R). R is calculated for each probe pair and compared to a predefined threshold, Tau. Probe pairs with R higher than Tau vote “present” and the voting result is summarized as a p-value. The greater the number of discrimination scores (R) that are above Tau, the smaller the p-value and the more likely the given transcript is truly present in the sample. Only probe sets with a “present” call in all three replicates of at least one treatment was considered to be “expressed”.
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