6 protocols
normalization data transformation protocol
GCOS v1.3 ID_REF = VALUE = MAS 5.0 signal ABS_CALL = indicating whether the transcript was present (P), absent (A), or marginal (M) DETECTION P-VALUE =
array scanning protocol
The scanning procedures were performed at the Partners HealthCare Center for Personalized Genetic Medicine (Cambridge, MA) with a GeneChip® Model 3000 7G scanner using the Affymetrix GCOS v1.3 operating system.
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).
labelling protocol
The TURBO Biotin labeling™ kit (end-labeling; Life Technologies Corporation) was used to label the aRNA obtained from amplified samples (15 μg).
nucleic acid extraction protocol
RNA extraction and isolation was performed using the Picopure™ RNA Isolation kit (Life Technologies Corporation), with a DNase step (Qiagen Inc., CA). The RNA underwent two rounds of linear amplification using the RiboAmp® kit (Life Technologies, CA). A dilution of the resulting aRNA (approximately 250 ng/µl) was used to determine transcript lengths with the Experion StdSens Labchip (Bio-Rad, CA). The concentration and purity of RNA were determined by absorbance measurements at the optical density of A260 and A280, using a NanoDrop spectrophotometer (Thermo Scientific, DE).
sample treatment protocol
Liquid nitrogen vapor fresh-frozen blocks containing the superior temporal gyrus (STG) from subjects were cut on a cryostat at 8μm. PV neurons were identified with a rapid immunohistochemistry procedure. Briefly, mounted sections were incubated in an anti-PV antibody (1:10 dilution; mouse, Sigma-Aldrich, MO) for 7 minutes and in peroxidase AffiniPure donkey anti-mouse IgG secondary antibody (1:10 dilution; Jackson ImmunoResearch Laboratories, PA) for 7 minutes, together with an RNase Inhibitor (40U/µl; Roche, Basel). Neurons were ultimately visualized with the NovaRED™ substrate-chromogen (12 minutes; Vector, CA). Approximately 300 PV-immunolabeled neurons per subject were captured onto a CapSure HS™ LCM cap (Life Technologies, NY).