gender: female age: 55 post mortem interval (pmi): 22 tissue source: brain tissue: superior temporal gyrus (STG) cell type: pyramidal cells in layer III of the STG
Treatment protocol
Liquid nitrogen vapor fresh-frozen blocks containing the superior temporal gyrus (STG) from subjects were cut on a cryostat at 8μm. These sections were stained with the Histogene™ quick staining kit (Life Technologies Corporation, CA) and identified in layer 3 of the STG strictly according to the following criteria: (1) cells with pyramidal morphology and (2) clearly identifiable proximal apical and basal dendrites. Approximately 500 neurons per subject were extracted from the surrounding tissue via laser capture microdissection (Arcturus XT™ system, Life Technologies Corporation, CA).
Extracted molecule
total RNA
Extraction protocol
RNA extraction and isolation was performed using the Picopure™ RNA Isolation kit (Life Technologies Corporation), with a DNase step (Qiagen Inc., CA). This typically resulted in approximately 1-25 ng of total RNA. Total RNA integrity was evaluated by inspection of the 18S/28S peaks of the electropherograms and virtual gel generated by Experion™ HighSens LabChip (Bio-Rad, CA) and the RNA Quality Indicator (RQI). If there was evidence of significant degradation, as indicated by a large area under the curve or if the peaks were not visible, the cells were re-captured from tissue sections stored at -80°C. The extracted RNA underwent two rounds of linear amplification using the RiboAmp® kit (Life Technologies Corporation, CA). A dilution of the resulting aRNA (approximately 250 ng/µl) was used to determine the mRNA transcript length with the StdSens Labchip (Experion, Bio-Rad, CA). The concentration and purity were determined by NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE) analysis at an optical density of A260 and A280. Only samples with transcript lengths extending past 600 nucleotides (eFigure 1) and an optical density ratio of higher than 1.8 were labeled for gene expression profiling
Label
biotin
Label protocol
The TURBO Biotin labeling™ kit (end-labeling; Life Technologies Corporation) was used to label the aRNA obtained from amplified samples (~15 μg).
Hybridization protocol
Labeled aRNA underwent hybridization as explained in the TURBO Biotin labeling™ kit (end-labeling; Life Technologies Corporation). Gene expression profiling was performed using the Affymetrix Human X3P GeneChip®, which possesses an extreme 3' bias in its probe design and hence is particularly suitable for samples that are prone to RNA degradation, such as postmortem human brain tissue. The hybridization and scanning procedures were performed at the Partners HealthCare Center for Personalized Genetic Medicine (Cambridge, MA).
Scan protocol
The scanning procedures were performed at the Partners HealthCare Center for Personalized Genetic Medicine (Cambridge, MA).
Description
S4 Used for analysis
Data processing
Each array was scanned twice and the Affymetrix Microarray Suite 5.1 software averaged the two images to compute an intensity value for each probe cell within each probe set. For the quality control step, we employed the dChip and Partek® software’s built-in function (Copyright, Partek Inc., St. Louis, MO). We then normalized all data with Partek’s standard normalization method (i.e. data has a mean of zero and a variance of one, and each column for each sample was divided by the average of all control samples). After a principal component analysis revealed the contribution of batch effect (scan date) to the observed variance, an Analysis of Covariance (ANCOVA) was performed with diagnosis and batch effect as covariates and the false discovery rate set at 10%. Differentially expressed genes were visualized by performing unsupervised hierarchical clustering as stringency of the filtering criteria (i.e. fold-change and p-value) was being varied to determine a representative gene list for pathway analyses. We employed three approaches to explore the biological significance of our expression data. First, differentially expressed genes were manually grouped to elucidate gene families that appeared to be dysregulated in schizophrenia. Second, pathway analyses were performed with two web-based algorithms, Ingenuity Pathway Analysis (Ingenuity® Systems) and MetaCore from GeneGo Inc. to map the differentially expressed genes onto biological functions and canonical pathways. With Ingenuity, the significance for each of the identified pathways was determined via a Fisher’s exact test, whereas GeneGo makes use of their algorithm for hypergeometric distribution, identifying pathways overrepresented with significant genes. Third, literature mining was performed to elucidate which of these pathways or gene families might be particularly pertinent for pyramidal neuronal functions and dendritic/synaptic architecture and plasticity. We also made note of differentially expressed genes that have been implicated as schizophrenia risk genes.
