Tissue: Logging slash of Jeffrey pine (Pinus jeffreyi Grev. & Balf.) infested with Ips pini were obtained from Ward Creek CA (39*08'41" N, 120*12'01" W, 2048 m) and placed in a greenhouse to allow emergence of adult beetles (Browne, J. Econ. Entomol. 65, 1499-1501, 1972). Upon emergence, beetles were typed and sexed and kept at 4°C on moist paper towels. Beetles were immobilized by chilling and then treated topically on the ventral surface of the abdomen with either 10 µg (±) juvenile hormone III (Sigma) in 0.5 µL acetone or acetone (control). They were then incubated in the dark at room temperature in groups of 10 in 60 mL plastic containers (Dixie) for specified times. At the end of the incubation, beetles were immersed in water and the anterior midguts excised under a stereo microscope. Midguts were gently purged of their contents and then immediately frozen in liquid nitrogen and stored at -80°C. Each treatment/control time point pair was replicated four times and each replicate contained tissue from 60 midguts (30 each for treatment and control). Treatment and control beetles were cohorts for a replicate pair and were incubated simultaneously. Beetles in different replicate pairs and time points were not always cohorts. Hybridizations: Hybridizations were done in quadruplicate, each with tissue from different groups of beetles. Total RNA was extracted using QIAshredder and RNeasy Mini kits (Qiagen) and quantified spectrophotometrically. Total RNA (approx. 40 µg) was reverse transcribed with oligo(dT)15 (Promega), Superscript III RT- (Invitrogen), and dNTPs (Promega) containing aminoallyl-dUTP (Sigma, aminoallyl-dUTP to dTTP ratio of 2 to 3). After alkaline hydrolysis of the RNA, clean up with a Microcon-30 (Millipore), and lyophilization, the aminoallyl-dUTP cDNA was reacted with Cy3 or Cy5 monofunctional reactive dyes (Amersham). Cy3 and Cy5 dyes were swapped equally between treatment and control samples among replicates (see source info for dye used for each target). After 60 min, unreacted dyes were removed from the labeled cDNA by a QIAquick spin column (Qiagen). The fluorescently labeled cDNAs from treatment and control samples were combined together with 20 µg herring sperm DNA (Promega) and 20 µg polydA (Amersham), lyophilized, and then resuspended in 20 µL hybridization buffer (50% formamide, 5x SSC, and 0.1% SDS). The hybridization mixture was denatured at 95°C for 5 min, centrifuged at 16,000 g for 5 min, and then immediately applied to the microarray preheated to 42°C. Prior to hybridization, microarrays were washed 5 min in 0.1% SDS, 5 min in 2x SSC, denatured 3 min in boiling water, placed into ice-cold ethanol, dried by centrifugation, incubated for 60 min at 42°C in pre-hybridization buffer (5x SSC, 0.1% SDS, and 1% BSA), washed 5 min in water, 5 min in 2-propanol, and then dried by centrifugation. The hybridization mixture was covered with a HybriSlip (22 x 40 mm, Sigma) and then the microarray was immediately placed into a preheated, humidified hybridization chamber and placed in a 42°C oven for 16-20 h. Microarrays were removed from the hybridization chamber and immediately washed with agitation at room temperature for 5 min each in 2x SSC with 0.03% SDS, 1x SSC, and 0.2x SSC, and then dried by centrifugation. Analysis: Arrays were scanned at 5 µm resolution with a Packard Bioscience ScanArray 4000 array scanner and analyzed with ScanArray and QuantArray software (versions 3.1 and 3.0 respectively). Median signal (adaptive spot algorithm) and background intensities were obtained for both dyes for each element. Data were background subtracted, globally and locally (across microarray surface, span=0.5, trim=0.1) normalized, log transformed (base 2), converted to mean log (intensities) and log (ratios), and then locally normalized across element signal intensity (span=0.7, trim=0.1) using online SNOMAD tools (Colantuoni et al., Bioinformatics 18, 1540-1, 2002). "Empty" and "Spotting Soln" elements were excluded from analysis by SNOMAD. Data for these two element types were arbitrarily filled with zeros to upload into NCBI GEO. To account for outliers, the elements in triplicate on the 4 replicated arrays were treated as unique measurements and a Q-test (96% confidence, Dean and Dixon, Anal. Chem. 23, 636-8, 1951) was performed on the 12 measurements. If a measurement tested as an outlier, its VALUE was replaced with the average VALUE of the two other replicate elements on the same array. At most, only 1 of the 12 measurements was excluded. Keywords = North American pine engraver beetle, anterior midgut, juvenile hormone, pheromone biosynthesis, Coleoptera, Scolytidae
Log (base 2) of channel 2 (treatment)/channel 1 (control). Equivalent to SNOMAD_logratiores except for outlier elements (see description), in which case it is the mean SNOMAD_logratiores of the other two replicate elements on the same array
