Phenol method with carbohydrate-enriched tissue such as tubers and trizol for other tissues such as leaves (SGED_SOP_3.1.1 + SGED_SOP_3.3.1)
Label
Cy3
Label protocol
Amino allyl labeling and direct labeling (SGED_SOP_5.1.1 + SGED_SOP_6.1.1)
Hybridization protocol
Hybridization with indirectly labeled mRNA (SGED_SOP_6.1.1, SGED_SOP_6.2.1-4)
Scan protocol
Axon 4000B scanner. Both the 635nm (red, Cy5) and 532nm (green, Cy3) channels are scanned simultaneously at 100% laser power, the PMT set between 600 and 950. Slides are scanned at a resolution of 10micron
Description
Study Description All plants used in this study were derived from the IBM 94 (Lee, Sharopova et al. 2002) mapping population and names used in the MIAME information correspond to Stapleton laboratory names. A conversion table identifying the official IBM names is presented below. Maize IBM94 seed was supplied by the Maize Coop and increased at the field nursery in Clayton, NC. For sowing, twelve seeds were placed in six-pack pots filled with sterilized vermiculite. Flats filled with 36 pots were placed in the greenhouse at the University of North Carolina Wilmington with solar illumination and daily watering until the majority of the plants were at the three-leaf stage (8 days). These growth conditions are similar to those used for previous inbred comparisons (Blum, Casati et al. 2004). Glass does not transmit UV-B, therefore, the plants received none of this radiation unless treated as part of the experimental design. For treatment, one set of flats with a six-pot of each line was moved onto irradiation benches with UV313 bulbs (Q-Panel Lab Products, Cleveland, OH USA) mounted 30 cm above the bench and covered with cellulose diacetate (US Plastics, Lima OH, USA). These plants received UV-B at a rate of 0.024 Wm-2, as previously described (Blum et al., 2004) for two hours (11 AM to 1PM) in August 2004. Control plants were placed under UV313 bulbs that were covered with plastic (MylarD, US Plastics, Lima, OH, USA) to block all UV-B radiation. Immediately following irradiation plants were removed from irradiation benches, and both control and experimental groups were allowed to recover for 3 h in the greenhouse. Three hours of recovery allows time for post-transcriptional regulation (such as RNA processing and small RNA cleavage) so that we are measuring the steady-state level of mRNA for each gene. Plants were irradiated centered on solar noon to maximize the amount of sunlight reaching the plants. Following recovery, the second and third seedling leaves of each of the control and experimental plants were harvested and immediately frozen in liquid nitrogen. The second and third replicate sets of plants were irradiated and harvested on succeeding days. Plant tissue batches used for RNA preparation were later used to verify genotypes. Genomic DNA was prepared from crushed tissue samples and PCR products from eleven SSR primers were scored by comparison to parental B73 and Mo17 PCR product sizes. The eleven SSR primers were chosen using and algorithm designed by T. Hudson, Dept. of Computer Science, UNCW; the algorithm allows rapid selection of a minimal combination of markers that will distinguish each IBM94 genotype. Total RNA was extracted from frozen tissue using Trizol (Invitrogen Co., Carlsbad, CA). After extraction, RNA was shipped on dry ice to the University of Arizona. Target labeling via RNA amplification and hybridization were in accordance to standard protocols posted on the Maize Microarray Project website (http://www.maizearray.org/maize_protocols.shtml ). Briefly, the target labeling protocol utilizes the Ambion Message Amp II kit to produce cRNA which is indirectly labeled using a dye coupling reaction. All arrays are prehybridized in 1% BSA, 5X SSC, 0.1% SDS. The resulting labeled cRNA (3 ug/slide) is hybridized to the arrays using a 50% formamide, 5X SSC, 0.1% SDS hybridization buffer at 42 C overnight and the arrays are washed using a series of high stringency washes. Official IBM ID Stapleton Laboratory ID Mo005 M2 Mo014 M7 Mo016 M9 Mo017 M10 Mo021 M11 Mo033 M22 Mo034 M23 Mo045 M29 Mo057 M36 Mo060 M38 Mo067 M43 Mo077 M47 Mo265 M51 Mo267 M53 Mo317 M69 Mo325 M73 Mo341 M77 Mo344 M78 Mo352 M81 Mo355 M83 Mo364 M86 Mo365 M87 Mo368 M88 Mo369 M89 Mo383 M55(akaM93)
Data processing
The TIFF images were quantified using Genepix 5.0. Local background was subtracted from the signal value and the data was normalized using the quantile method in the limma package of bioconductor.
Background (CH2_BACKGROUND) subtracted raw intensity (F532 Mean - B532 Media)
CH2_BACKGROUND
CH2 background median intensity (B532 Media)
FLAG
B: no flag, good spot; X: undetectable spot; M: flagged for diameter < 70 microns, the percentage of saturated pixels > 30% or not validated PCR product
