Affymetrix CEL files generated after hybridization of samples to MouseExon 1.0 ST arrays were imported into Partek® Genomics Suite software package, version 6.2, build 6.06.0517. Copyright © 2007 ( Partek Inc., St. Louis, MO, USA.). Microarray data analysis was performed using the predefined workflow for exon analysis.
Only 193387 core metaprobes set which consisted of probes targeting exons with RefSeq mRNA evidence are regarded as the most confident were analyzed.
First, GCRMA (Robust Multi-array analysis with correction for GC content) algorithm was used to background correct the data. Subsequently, the data were normalized using quantile normalization and per probe normalization was applied by using the mean of each probe .
The slides were scanned using the GeneChip Scanner 3000450 (Affymetrix. The scanner operating software, GCOS, converted the signal on the chip into a DAT file, which was used for generating CEL files for analysis.
(Parameters: Scanning hardware = OTHER: 3000450 (Affymetrix), Scanning software = Scanning software)
Manufacturer's protocol was strictly adhered. Online copy of the protocol can be obtained at the following address:
(Parameters: Chamber type = OTHER: Innova™ 4080 shaking incubator, Quantity of label target used = 10, Mass unit = Micro gram, time = 18, Tiny time unit = hours, Volume = 260, Volume unit = Micro litre, temperature = 37)
The microarray experiment was carried out by Australian Genome Research Facility (AGRF). Manufacturer’s protocol and Affymetrix microarray protocol were strictly adhered.
In brief, 3 ug of RNA was subjected to rRNA removal procedure using RiboMinus Human/Mouse Transcriptome Isolation Kit ( Invitrogen) and subsequently labelled using the Affymetrix WT cDNA Amplification kit (Millenium Sciences). The sense cDNA was fragmented using the APE1 ( apurinic/ apyrimidic endonuclease 1) and UDG (uracil DNA glycosylase) and quality checked using the Agilent Bioanalyser 2100 using the NanoChip protocol.
The fragmented single stranded cDNA was end-labelled using terminal deoxynucleotidyl transferase enzyme from the WT Terminal Labelling kit (Millenium Sciences). A total of 5 ug of labelled cDNA was then hybridised to the MouseExon 1.0 ST Array GeneChip (Millenium Sciences).
(Parameters: Amplification = none, Mass unit = Micro gram)
For in utero atRA treatment pregnant female GR+/- mice were injected subcutaneously with either all-trans retinoic acid (atRA, 2mg/kg in peanut oil) or vehicle alone (peanut oil) on E16.5, E17.5 and E18.5. Three hours after the final injection pregnant females were euthanised and fetuses collected by caesarian section. Fetal lungs were removed and the left lung was collected for histological analysis while the right lung was collected for RNA extraction. Genotyping was performed as described previously (1) and samples were organized in four groups; untreated GR+/+ (n=6), atRA treated GR+/+ (n=9), untreated GR-/- (n=4) and atRA treated GR-/- (n=5).
(1) Cole TJ, Harris HJ, Hoong I, Solomon N, Smith R, Krozowski Z, Fullerton MJ 1999 The glucocorticoid receptor is essential for maintaining basal and dexamethasone-induced repression of the murine corticosteroid-binding globulin gene. Mol Cell Endocrinol 154:29-36
Isogenic C57BL/6 GR+/- mice were obtained by backcrossing 129/sv-C57BL/6 mixed background GR+/- mice more than 10 times (1) to generate GR+/- mice on an isogenic C57BL/6 background. GR+/- Timed matings of isogenic C57BL/6 GR+/- mice were performed and fetuses collected by caesarian section at E18.5. Fetuses were genotyped as previously described (2). All animal experimentation was approved by the School of Biomedical Sciences Animal Ethics Committee, Monash University.
(1)Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, Aguzzi A, Fantuzzi G, Hummler E, Unsicker K, Schutz G 1995 Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev 9:1608-1621
(2)Cole TJ, Harris HJ, Hoong I, Solomon N, Smith R, Krozowski Z, Fullerton MJ 1999 The glucocorticoid receptor is essential for maintaining basal and dexamethasone-induced repression of the murine corticosteroid-binding globulin gene. Mol Cell Endocrinol 154:29-36
(Parameters: time unit = days, temperature unit = C)
Total RNA was extracted from distal lung tissue using the TRIzol® Plus RNA Purification System (Invitrogen) according to the manufacturer’s instructions. Briefly, the frozen tissue was homogenised in TRIzol reagent, precipitated using isopropanol and the RNA pellet washed in ethanol. Total RNA was finally dissolved in nuclease-free H2O and yield and quality of RNA was determined using standard UV spectrophotometry and gel electrophoresis.
(Parameters: Extracted product = total_RNA, Amplification = none)