tissue: prefrontal cortex cohort: intraoperative-2 diagnosis: ET age (yr): 61 Sex: M
Extracted molecule
total RNA
Extraction protocol
Intraoperative tissue was collected during previously scheduled DBS implantation surgery. All PD patients were off dopaminergic medication for the surgery. Since DBS electrode insertion requires a cortical incision, no additional cortical tissue was disrupted for the patients in this study. A standard coronal burr hole placement for usual clinical DBS trajectory and targeting was utilized. These were ~14-mm diameter holes, typically located over the middle frontal gyrus or rarely located over the lateral aspect of the superior frontal gyrus (i.e., Brodmann areas 8, 9/46). After dural opening, a corticotomy at the DBS insertion point was made bilaterally under direct vision to avoid visible blood vessels. Through this opening, ~2-mm cupped biopsy forceps (~the same diameter as a DBS insertion cannula) were used to grasp a small biopsy (~5 mg) from directly beneath the pial surface (Figure 1). The collected tissue was immediately passed off the sterile field and frozen in the operating room using dry ice. Samples were collected separately from the left and right hemispheres and pooled together for RNA extraction and downstream analysis. Due to the brevity of tissue collection, no meaningful additional time was added to the surgery. One to three 2-mm diameter stainless steel DBS implantation cannulas were then passed through the same cortical opening, and surgery commenced in the usual manner. No immediate or delayed complications from the tissue collection procedure were observed. To compare gene expression profiles from our intraoperative tissue specimens with prior data, we collected postmortem samples from the superior/middle frontal gyrus (BA 8, 9/46), the same frontal region targeted in DBS patients. All postmortem frontal cortex tissue was obtained from the Iowa NeuroBank Core (https://medicine.uiowa.edu/inbc/), with informed consent forms signed by the next of kin as required by state law. The postmortem cases were PD patients or neurotypical control patients available for study without PD or ET.
Label
n/a
Label protocol
Because of the challenge of intraoperative tissue, comparisons between intraoperative and postmortem cohorts, and small samples, we chose the NanoString nCounter Human Neuropathology Panels. Gene expression profiles of intraoperative tissue collected during DBS-implantation surgery and postmortem frontal tissue were generated using identical NanoString nCounter Human Neuropathology Panels (NanoString Technologies, Inc., Seattle, WA). Human tissue samples were submitted to the Iowa NeuroBank Core in the Iowa Neuroscience Institute. Total RNA (100 ng per sample) was extracted using the RNeasy Plus Mini Kit (Qiagen, Germantown, MD) following the manufacturer’s protocol. The quality and concentration of RNA samples were quantified using NanoDrop (Thermo Scientific, Waltham, MA), Fragment Analyzer (Agilent, Clara, CA), and Bioanalyzer (Agilent) systems in the Iowa Neuroscience Institute and in the Iowa Institute of Human Genetics, Genomics Division. Samples with a Bioanalyzer RNA integrity number (an index of RNA quality control) of >3 were analyzed with the NanoString nCounter Human Neuropathology Panel and Custom CodeSet (C8574, C9676) assays, designed in consultation with the NanoString bioinformatics team. All intraoperative mRNA assays were performed on the NanoString nCounter SPRINT Profiler for image acquisition and data processing, without amplification or generation of cDNA (Geiss et al 2008). Due to instrumentation updates, the postmortem assays were performed using the NanoString nCounter MAX system, according to the manufacturer’s instructions. Gene expression profiles for a total of 780 genes were screened using target-specific biotinylated probes and barcoded reporter probes in the NanoString nCounter neurodegenerative cartridge.
Hybridization protocol
N/A
Scan protocol
N/A
Data processing
Reporter Code Count (RCC) and Reporter Library Files (RLF) generated by the nCounter SPRINT Profiler were imported into NanoString nSolver 4.0 Advanced Analysis software, version 2.0.134. The manufacturer’s recommended default parameters were used for imaging, quality control flagging, binding density, positive control linearity, and positive control limit of detection. Each sample in the intraoperative and postmortem cohorts was first normalized to the geometric mean determined by the six External RNA Controls Consortium (ERCC) synthetic RNA Spike-In Controls designated as positive controls (PosA to PosF), with default flagging of normalization factors (<0.3 and >3). These ERCC RNA Spike-In Controls were used both within each experiment and across all the nCounter experiments to create a standard baseline measurement of RNA samples across various samples and batches. We then applied normalization using the geometric mean of custom housekeeping genes, with default flagging of normalization factors (<0.1 and >10). Thirteen stably expressed housekeeping genes were selected for the intraoperative samples: CCDC127; UBE2D2; ASB7; LARS; MTO1; CSNK2A2; TADA2B; SUPT7L; CNOT10; AARS; FAM104A; CYC1; and RPL13. The geometric mean of the two most stable housekeeping genes, CYC1 and UBE2D2 were selected for normalization in the postmortem cohort. Normalized gene expression datasets were assessed using fold changes, p values, and enriched pathways.
