strain: C57BL/6J gender: Female surgery: ovariectomy stress: stress age: 17-week-old tissue: medial prefrontal cortex individual: same as WB_OVX+stress_1
Treatment protocol
Mouse blood (300 µl) was collected under pentobarbital anesthesia (50 mg/kg, i.p.) via the vena cava. The blood was heparinized immediately and centrifuged at 1,000 × g for 2 min. The pellet was dissolved in ice-cold lysis buffer containing 2-mercaptoethanol. Immediately after the blood was drawn, the mouse was decapitated, and the brain was removed. The decapitation was completed within 5 min of the anesthesia taking effect to minimize the effect of pentobarbital on gene expression. Coronal slices (1 mm thickness) were sectioned using a brain slicer, and the mPFC was dissected under a stereoscopic microscope. The dissected tissues were immersed in the RNA stabilization solution RNAlater (Qiagen K.K., Tokyo, Japan) and stored until RNA extraction.
Extracted molecule
total RNA
Extraction protocol
The total RNA in the blood lysates was extracted using a GeneJet Whole Blood RNA Purification Mini Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA from the mPFC tissues was extracted using an RNeasy Micro Kit (Qiagen K.K.) according to the manufacturer’s instructions. The RNA quantity and quality were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc.) and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) as recommended.
Label
Cy3
Label protocol
Total RNA was amplified and labeled with Cyanine 3 (Cy3) using a one-color Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Briefly, 100 ng of total RNA was reverse-transcribed to obtain double-stranded cDNA using a poly dT-T7 promoter primer. The primer, template RNA, and quality-control transcripts of known concentrations and quality were first denatured at 65°C for 10 min and then incubated for 2 h at 40°C with 5× first-strand buffer, 0.1 M dithiotreitol, 10 mM dNTP mix, and AffinityScript RNase Block Mix. The AffinityScript enzyme was then inactivated at 70°C for 15 min. The cDNA products were used as templates for in vitro transcription to generate fluorescent cRNA. The cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3-labeled CTP and then incubated at 40°C for 2 h. The labeled cRNAs were purified using Qiagen’s RNeasy mini spin columns and eluted using 30 µl of nuclease-free water. After cRNA was amplified and labeled, the cRNA quantity and cyanine incorporation were determined using a NanoDrop ND-1000 spectrophotometer and an Agilent Bioanalyzer.
Hybridization protocol
For each hybridization, 600 ng of Cy3-labeled cRNA was fragmented and hybridized at 65°C for 17 h to an Agilent SurePrint G3 Mouse GE 8×60K Microarray (Design ID: 028005).
Scan protocol
After washing, the microarrays were scanned using an Agilent DNA microarray scanner.
Description
a pair of WB_OVX+stress_1
Data processing
The intensity values of each scanned feature were quantified using Agilent feature extraction software version 10.7.3.1, which performs background subtractions. The normalization was performed using Agilent GeneSpring GX version 11.0.2 (per chip: normalization to the 75th percentile shift; per gene: none). The probes that were declared as “present” in all the assayed samples and that displayed a raw intensity value above 50 in at least 2 samples were used for the following statistical analyses.
