tissue: ventrolateral prefrontal cortex (PFC) diagnosis: major depressive disorder gender: male population: French-Canadian age: 36 ph: 6.54 rin: 5.6
Treatment protocol
Post-mortem prefrontal cortex (BA44) brain tissue was obtained in collaboration with the Quebec Coroner's Office and the Suicide section of the Douglas-Bell Canada Brain Bank (Douglas Mental Health University Institute, Montreal, Quebec, Canada). A total of 25 brain samples were included in the present study. All individuals were of French-Canadian origin, a homogeneous population with a well-documented founder effect, and were matched for refrigeration delay, age and pH. Refrigeration delay refers to the difference between the estimated time of death (determined by the pathologist through external body examination details) and the time at which the body was refrigerated. Psychological autopsies were performed post-mortem on both cases and controls by a panel of psychiatrists and diagnoses were assigned based on DSM-IV criteria. The control group had no history of suicidal behavior or major mood or psychotic disorders.
Extracted molecule
total RNA
Extraction protocol
Total RNA (including miRNA fraction) was isolated from frozen brain using the miRNeasy Mini Kit protocol (Qiagen, Canada) with no modifications. RNA and miRNA yield and quality were determined using the Nanodrop 1000 (Thermo Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA), respectively.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the microRNA complete labeling and hybridization kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification. This method involves the ligation of one Cyanine 3-pCp molecule to the 3' end of a RNA molecule with greater than 90% efficiency. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
Samples were dried using a vacuum concentrator at 50°C on the medium-high heat setting for one hour. All samples were resuspended in 17 uL of nuclease-free water, 1 uL of Hyb spike-in solution, 4.5 uL of 10X GE Blocking agent and 22.5 uL of 2X Hi-RPM Hybridization Buffer , incubated at 100°C for 5 minutes followed by a 5 minute ice water bath, as suggested by the manufacturer's instructions. Samples were hybridized on Agilent-021827 slides at 55°C and rotated at 20rpm for 20 hours. After hybridization, microarrays were washed at room temperature with GE Wash Buffer 1 (warmed to 37°C, Agilent) and GE Wash Buffer 2 (warmed to 37°C, Agilent) ), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565C) using one color scan setting on the Unrestricted Human miRNA microarray V3 (Agilent Design ID: 021827; Product Number: G4470C). Format 8x15K, Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR low 5%.
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. We used the AgiMicroRna package in Bioconductor to summarize the data followed by quantile normalization.
