Computational protocol: Novel Application of Stem Cell-Derived Neurons to Evaluate the Time- and Dose-Dependent Progression of Excitotoxic Injury

Similar protocols

Protocol publication

[…] RNA was extracted from ESNs using the RNeasy Mini Kit (Qiagen, Germantown, MD) and RNA quality was assessed using the NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific). Expression profiling using next-generation sequencing (RNAseq) was conducted as previously described , . Reads were assembled, annotated and screened for differential expression of Gria2 isoforms using the Seqman Pro software package (DNAStar, Inc, Madison, WI). Library sizes were normalized using the DESeq normalizedCounts function and normalized reads are presented as pseudocounts.For quantitative, real-time reverse-transcription PCR (QPCR), cDNA was synthesized using the RT2 First Strand Kit (SaBiosciences, Frederick, MD) according to manufacturer's protocol). QPCR was performed using a BioRad-CFX 96-well Real-Time System with iQ SYBRGreen Supermix in a PTC-100 MJ Research thermal cycler (Waltham, MA). Primer sequences were from the Harvard University Medical School Primer Bank () . PCR conditions were 95°C for 3 min, followed by 39 cycles of 95°C for 10 sec, 54°C for 10 sec, and 72°C for 30 sec, then a melt curve from 54 to 95°C in 0.5°C increments. The ΔCT for control and treated samples were normalized to β3-tubulin to determine the normalized log2 fold change. […]

Pipeline specifications

Software tools DNASTAR Molecular Biology Suite, DESeq
Application RNA-seq analysis
Organisms Mus musculus
Diseases Wounds and Injuries, Neurotoxicity Syndromes, Drug-Related Side Effects and Adverse Reactions