tissue: Fetal brain brain region: Hypothalamus developmental age: 145 days of fetal age
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from each cortex, hippocampus, hypothalamus and brainstem using Trizol (Invitrogen, Carlsbad, CA) and following the manufacturer’s directions. The RNA was resuspended in RNAsecure, and stored at -80ºC in aliquots until use. For microarray analysis 20 ug of these RNAs, were DNase treated using the Turbo RNase-free DNase kit (Ambion, Foster City, CA), the concentration determined with a Nanodrop spectrophotometer (ND-1000, ThermoFisher, Wilmington DE) and the integrity of the RNA was measured using an Agilent Bioanalyzer, 2100 model.
Label
Cy3
Label protocol
One µg of the DNase-treated RNA was labeled with Cyanine 3 (Cy3) CTP with the Agilent Quick Amp kit (5190-0442, New Castle, DE) according to their methodology, purified with the Qiagen RNeasy kit (Valencia, CA) according to Agilent’s revision of the Qiagen protocol as shown in the Quick Amp kit protocol except that the microcentrifugation spins were performed at room temperature instead of 4ºC. The resulting labeled cRNA was analyzed with the Nanodrop spectrophotometer, and the specific activities and the yields of the cRNAs were calculated. The labeled cRNA was stored at -80ºC until use.
Hybridization protocol
600 ng of each labeled cRNA was fragmented and then mixed with hybridization buffer using the Agilent gene expression hybridization kit. These were applied to a sheep 8 X 15 K array slide (Agilent 019921), containing 8 arrays with 15,208 oligomers with a length of 60 bases corresponding to 15,208 ovine genetic sequences and hybridized at 65 °C for 17 h at 10 rpm. After hybridization, the slides were washed for 1 minute at room temperature with Wash Buffer 1 (Agilent) and for 1 minute with 37 °C Wash buffer 2 (Agilent). After dry the excess of liquid, the slides were washed for 30 seconds in the stabilization and drying solution.
Scan protocol
The slides were scanned with an Agilent 2-dye G2505B scanner that is compatible with any 1' x 3' glass slide.
Description
Gene expression in fetal hypothalamus at 145 days of gestation-Replicate 4
Data processing
Features were extracted with Agilent Feature extraction 9.1 software. The limma package was employed to import the raw data into R (http://www.r-project.org), perform background correction and normalize the data by the quantile normalization method. Control probes and low expressed probes were filtered out, retaining for further analysis the probes that were at least 10% brighter than the negative controls on at least four arrays.
