Gene Expression

The grey matter trimmed from the rostral-caudal 1/3 of the fresh frozen middle frontal gyrus was used. This tissue was pulverized, and the total RNA was extracted using TRIZOL. Total RNA was extracted from all six layers cortical layers of the middle frontal gyrus, at the level anterior to the genu of the corpus callosum, not including the frontal pole. White matter was removed from each block of tissue with a scalpel or razor blade before RNA extraction.

Gene expression changes for the cluster of these seven genes were confirmed with quantitative real-time polymerase chain reaction (qPCR) using an ABI Prism 7900HT Fast Real Time PCR system with a 384-well format for both targets and housekeeper normalizing controls (Applied Biosystems, Foster City, CA, USA) as previously reported^{10, 30}. Specifically, pre-designed Taqman gene expression assays (Applied Biosystems, Foster City, CA, USA) were used to measure the following transcripts: SERPINA3 (Hs003153674_m1), IL6 (Hs00174131_m1), IL6ST (Hs01006741_m1), IL8 (HS00174103_m1), IL1B (Hs01555410_m1), NFKB1 (Hs00765730_m1), and PTGS2 (Hs00153133_m1). ACTB (Hs99999903_m1), GAPDH (Hs99999905_m1), TBP (Hs00427621_m1) and UBC (Hs00824723_m1) were used as normalizing transcripts and did not differ according to experimental groups.

Using a two-step recursive cluster analysis, we found that four of these seven transcripts (SERPINA3, IL-6, IL-8, and IL-1β) contributed significantly to the model and were thus used to define the ‘High’ vs ‘Low’ neuroinflammation groups¹¹. Missing and outlier values were replaced using the SPSS-derived expectation maximization algorithm in order to retain as many cases as possible. The clustering was performed on all cases, and in order for the model quality to be considered acceptable, it had to exceed 0.5 on a scale of 0 to 1.0. Additionally, the individual predictor importance for each transcript had to exceed 0.4 on the same scale.

For the current study, we clustered a subset of the postmortem samples with both CMV antibody data and gene expression data (n=82; 30 schizophrenia, 23 BD, and 29 controls) into “high” (n=30, 13 schizophrenia, 8 BD, and 9 controls) or “low” inflammation groups (n=52, 17 schizophrenia, 15 BD, and 20 controls) using the identical methodology described above.