This study was conducted using postmortem human brain specimens from tissue collections at the Department of Neurobiology at Yale University School of Medicine and the Clinical Brain Disorders Branch of the National Institute of Mental Health. Additional specimens were procured from the Human Fetal Tissue Repository at the Albert Einstein College of Medicine (AECOM), the Brain and Tissue Bank for Developmental Disorders at the University of Maryland, the Birth Defects Research Laboratory at the University of Washington, Advanced Bioscience Resources Inc. and the MRC-Wellcome Trust Human Developmental Biology Resource at the Institute of Human Genetics, University of Newcastle, UK. Tissue was collected after obtaining parental or next of kin consent and with approval by the institutional review boards at the Yale University School of Medicine, the National Institutes of Health, and at each institution from which tissue specimens were obtained. Specimens range in age from 5.7 post-conceptual weeks (PCW) to over 80 years. Of 57 post-mortem brain specimens included in this study, 24 were obtained with either left or right hemisphere, and 33 were obtained with both hemispheres. Embryonic and fetal age was extrapolated based on the date of the mother’s last menstruation, characteristics of the fetus noted upon ultrasonographic scanning, foot length of the fetus, and visual inspection.
Extracted molecule
total RNA
Extraction protocol
A bead mill homogenizer (Bullet Blender, Next Advance) was used to lyse the pulverized tissue. Each pulverized tissue sample was transferred to a chilled safe-lock microcentrifuge tube (Eppendorf). A mass of chilled stainless steel beads (Next Advance, cat# SSB14B) equal to the mass of the tissue was added to the tube. Two volumes of Buffer RLT (Qiagen) were added to the tissue and beads. Samples were mixed in the Bullet Blender for 1 min at a speed of six. Samples were visually inspected to confirm desired homogenization and then incubated at 37 °C for five min. Buffer RLT was added up to 0.6 ml, and samples were mixed in the Bullet Blender for 1 min. Total RNA was extracted using a non-phenolic procedure (RNeasy Plus Mini Kit, Qiagen), followed by DNase treatment (TURBO DNase, Ambion) as per manufacturers’ instructions. Optical density values of extracted RNA were measured using a NanoDrop (Thermo Scientific) to confirm an A260:A280 ratio above 1.9. RNA integrity number (RIN) was determined for each sample using Bioanalyzer RNA 6000 Nano Kit or Bioanalyzer RNA 6000 Pico Kit (Agilent), depending upon the total amount of RNA.
Label
Biotin
Label protocol
Exon array hybridizations were performed at the Yale Center for Genome Analysis and at Gene Logic Inc. (Gaithersburg, MD). Reverse transcription (RT) was performed to generate cDNA from total RNA using RT primers designed using an oligonucleotide matching algorithm. For the selective cDNA synthesis, the Ambion WT Expression kit (Ambion) was used in combination with the GenechipWT Terminal Labeling and Controls Kit (Affymetrix) for target preparation, according to manufacturer recommendations. PolyA controls were added to the input RNA to measure efficiency of target amplification. Fragmented and labelled second cycle cDNA (5.5 μg) was added to a hybridization cocktail prior to loading of 200 μl onto individual Affymetrix Human Exon 1.0 ST arrays.
Hybridization protocol
Microarrays were hybridized at 45 °C for 16–24 hours, washed and stained using an Affymetrix FS450 fluidics station, according to manufacturer recommendations. Microarrays were scanned on a GeneChip Scanner 3000 and visually inspected for hybridization artefacts. Exon chip analysis was performed using Affymetrix Power Tools 1.12.0. Probe level data was summarized into probe set level data using the Robust Multichip Average (RMA) background correction algorithm in combination with an R-script. The raw image files (.DAT files) were analyzed using Affymetrix GeneChip Operating Software to generate CEL files.
Scan protocol
Affymetrix Gene Chip Scanner 3000
Description
HSB102_A1C_R
Data processing
Partek Genomics Suite version 6.5 (Partek Incorporated, St. Louis, MO, USA) was used to normalize raw exon array data and to summarize expression of the probe set and transcript cluster. Affymetrix CEL files that passed QC analyses were imported into Partek Genomics Suite using the default Partek settings: RMA background correction, quantile normalization, mean probe set summarization, and log2 transformation. Only high-quality core probe sets, as defined by Affymetrix, were included because theyalways have reliable annotations from RefSeq (www.ncbi.nlm.nih.gov) or Ensembl (www.ensembl.org). It is noted that thedatasets were annotated according to the UCSC human genome hg19 reference sequence (http://genome.ucsc.edu/cgi-bin/hgGateway).Besides,105,271 core probes (within 62,448 prob sets) contained common SNPswas removed because SNP-containing probe sets may lead to reduced signal intensity and result in spurious results. These probes were defined in the probe group file HuEx-1_0-st-v2.r2-SNPs-Excluded.pgf provided by Affymetrix, which is basedon the dbSNPdatabase (version 129, April 2008) andSNPinprobe_1.0 database. Removal of SNP-affected probes was accomplished in Partek program.Probe sets and gene intensities was summarized only on those probes not affected by SNPs.Finally, a total of 17,565 genes (transcript clusters) were surveyed for downstream analysis. probe group file: HuEx-1_0-st-v2.r2-SNPs-Excluded.pgf meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps
