Chicken anterior pituitary gland cells, embryonic day 11
Biomaterial provider
Allen's Hatchery (Seaford, Delaware)
Growth protocol
Fertilized chicken eggs were placed in 37.5 C humidified incubator on embryonic day 0 and removed from the incubator on embryonic day 11. Pituitary glands from embryonic day 11 embryos were extracted and dispersed into individual cells by trypsin digestion and mechanical agitation. Dispersed pituitary cells were plated in poly-L-lysine coated 12-well culture plates in serum-free medium supplemented with 0.1% bovine serum albumen (BSA), 5 µg/ml bovine insulin, 5 µg/ml human transferrin, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate. Cells were allowed to attach for 1 h in a 37.5 °C, 5% CO2 atmosphere. Cells were then either 1) pretreated for 1.5 h with 10 microgram/ml cycloheximide and then subsequently cultured in the presence of corticosterone (10-9M) for 0, 1.5, 3, 6, 12, or 24 hrs, or (2) cultured with corticosterone (10-9M) with no cycloheximide pretreatment for 0, 1.5, 3, 6, 12, or 24 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA) and was amplifed using a modification of the Eberwine Procedure (Phillips J, Eberwine JH 1996 METHODS: A Companion to Methods in Enzymology 10: 283-288). Briefly, 500 ng was reverse-transcribed using SuperScript II (Invitrogen, Carlsbad, CA) and an oligo(dT) primer containing a T7 promoter site (5'-GGCCAGTGAATTGTAATATCGACTCACTATAGGGAGGCGGT24-3'; Affymetrix, Santa Clara, CA). Following second strand synthesis and purification, double-stranded cDNA was used as a template for in vitro transcription using the T7 MEGAscript kit (Ambion, Austin, TX) and the resulting amplified antisense RNA was quantified with the Ribogreen RNA Quantitation Kit (Invitrogen, Carlsbad, CA).??
Label
Cy3
Label protocol
1 microgram amplified antisense RNA (equivalent to 1 microgram mRNA) was converted to cDNA using random primers with the Amino Allyl cDNA Labeling Kit (Ambion), and Cy3-monoreactive NHS esters (Amersham Biosciences, Piscataway, NJ) were coupled to the cDNA. Labeled cDNA was purified from unicorporated dye using CyScribe GFX Purification Kit (Amersham Biosciences).
Channel 2
Source Name
Reference pool of chicken embryonic pituitary gland amplified RNA, embryonic day 11
Chicken embryonic pituitary amplified antisense RNA, pooled from all replicates (n=4) to be used as a reference sample (48 samples total)
Extracted molecule
total RNA
Extraction protocol
Extraction and amplification was conducted as described for channel 1 for each of the 48 samples used in the study, and a pool was made of 2 micrograms from each sample.
Label
Cy5
Label protocol
1 microgram amplified antisense RNA (equivalent to 1 microgram mRNA) from the pool was converted to cDNA using random primers with the Amino Allyl cDNA Labeling Kit (Ambion) and Cy5-monoreactive NHS esters (Amersham Biosciences, Piscataway, NJ) were coupled to the cDNA. Labeled cDNA was purified from unicorporated dye using CyScribe GFX Purification Kit (Amersham Biosciences) and a pool was made of all Cy5-labeled cDNA to be split among the 48 slides in the experiment.
Hybridization protocol
The microarray was hybridized overnight at 42 C with Cy3-labeled embryonic day 11 cDNA and an aliquot of the Cy5-labeled reference pool cDNA using microarray hybridization buffer (Amersham Biosciences). Slides were washed in increasing stringency using salt sodium citrate.
Scan protocol
The microarray slides were scanned using a 418 confocal laser scanner (Affymetrix) at 550 nM for Cy3 and 649 nM for Cy5 to generate two TIFF images for each slide.
Description
Chicken embryonic pituitary gland, embryonic day 11, replicate 4
Data processing
The two images from each slide were processed with Spotfinder version 2.2.4 (The Institute for Genomic Research, Rockville, MD) using the following settings: Otsu thresholding algorithm (minimum spot size 3 and maximum spot size 25); keep flagged values; keep raw data; and quality control filter set so 50% cutoff equals background plus 1 standard deviation. Data were normalized using Microarray Data Analysis System (MIDAS) version 2.18 (The Institute for Genomic Research). In MIDAS, channel A and B flags and channel A and B background checking were used, and the signal-to-noise threshold was set at 3.0. Data from the Cy3 channel were first Lowess (LocFit) normalized by pin (block) using a smoothing parameter of 0.33 with Cy5 as the reference and then sujected to standard deviation regularization first by pin (block) then by slide with Cy5 as the reference. The data from each spot on each slide were then expressed as log2-ratio, or log2(normalized Cy3/raw Cy5), which is given in the data table below. Log2-ratios for the spots were statistically analyzed by one-way analysis of variance using Statistical Analysis Systems version 8.02 (SAS Institute, Cary, NC) to identify cDNAs that were differentially expressed on at least one of the time points (n = 4; P < 0.05), and a fold-change of > 2.0 was used as a filter for false positives.
Characterization of Glucocorticoid-Induced Changes in Gene Expression in the Embryonic Pituitary Gland.
Data table header descriptions
ID_REF
ID in platform
VALUE
log2(normalized Cy3/raw Cy5) with flagged values removed
CH1_Normalized
Cy3 normalized intensity
CH1_RAW
Cy3 raw intensity
CH2_RAW
Cy5 raw intensity
CH1_Flag
Cy 3 flags; B (0-50 non-saturated pixels in spot) and C (> 50 non-saturated pixels in spot) denote satisfactory values; A (0 non-saturated pixels in spot), X (spot rejected due to shape and intensity relative to background), Y (background higher than spot intensity), and Z (spot not detected) denote unsatisfactory values
CH2_Flag
Cy 5 flags; B (0-50 non-saturated pixels in spot) and C (> 50 non-saturated pixels in spot) denote satisfactory values; A (0 non-saturated pixels in spot), X (spot rejected due to shape and intensity relative to background), Y (background higher than spot intensity), and Z (spot not detected) denote unsatisfactory values
Area
Spot area in pixels
Sat_factor
saturation factor
QCscore
average quality control score for both channels (Cy3 and Cy5)
