Computational protocol: Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers

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Protocol publication

[…] Total RNA was extracted from frozen cerebellum and frontal cortex using the RNeasy Plus mini kit (Qiagen), following manufacturer’s protocols. RNA quality and quantity were determined with an Agilent 2100 Bioanalyzer using the RNA Nano Chip (Agilent Technologies). High quality RNA was obtained from the cerebellum and frontal cortex of 49 and 48 patients, respectively. We performed a reverse transcription reaction with 300 ng of RNA as template, using an equal ratio of Random Hexamers and Oligo dT primers and the SuperScript III Kit (Invitrogen). Quantitative real-time PCR was then done on an ABI7900 PCR system (Applied Biosystems) using TaqMan gene expression assays (Life Technologies) in accordance with the manufacturer’s recommendations. We assessed neuronal markers synaptophysin (SYP; Hs00300531_m1) and microtubule-associated protein 2 (MAP2; Hs00258900_m1) as well as C9ORF72 transcript variant 3 (NM_001256054.2; Hs00948764_m1). Using R Statistical Software (version 3.2.0; R Foundation for Statistical Computing), we calculated the median of replicates and took the geometric mean of neuronal markers, and subsequently determined expression levels relative to control subjects utilizing the ΔΔ Ct method. For transcript variant 1 (NM_145005.6), digital molecular barcoding (NanoString Technologies) was performed by the manufacturer using 100 ng of RNA as template. The NanoStringNorm R package and nSolver Analysis Software (NanoString Technologies, version 2.5) were used to account for technical assay variation using the geometric mean method. Maximum background subtraction was then performed, and data was normalized to the geometric mean of neuronal markers SYP and MAP2. […]

Pipeline specifications

Software tools NanoStringNorm, nSolver Analysis Software
Application nCounter System
Organisms Homo sapiens
Diseases Amyotrophic Lateral Sclerosis, Cerebellar Diseases, Motor Neuron Disease, Frontotemporal Lobar Degeneration, Frontotemporal Dementia