Computational protocol: Deriving dopaminergic neurons for clinical use. A practical approach

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Protocol publication

[…] Total RNA was extracted from collected from duplicate sample pellets (RNeasy; Qiagen) according to the manufacturer's protocol. RNA quantity (Qubit RNA BR Assay Kits; Invitrogen) and quality (RNA6000 Nano Kit; Agilent) was determined to be optimal for each sample before further processing. 200 ng RNA per sample was amplified using the Illumina Total Prep RNA Amplification Kit according to manufacturer's protocol and quantified as above. 750 ng biotinylated RNA per sample was hybridized to Illumina HT-12v4 Expression BeadChips, scanned with an Illumina iScan Bead Array Scanner, and quality controlled in GenomeStudio and the lumi bioconductor package. All RNA processing and microarray hybridizations were performed according to manufacturer's protocols. In GenomeStudio, probes were filtered for those detected with a P value of 0.01 in at least one sample and exported for normalization in R. Raw probe expression values were transformed and normalized using the robust spline normalization (RSN) as implemented in the lumi R/Bioconductor package. To obtain the Venn Diagram, Qlucore Omni Explorer was used. Transcripts that were differentially expressed between pairs of cell types (hPSC vs Substantia Nigra, hPSC vs NSC, and hPSC vs Dopaminergic neurons) were identified using two-tailed Student's t-test with P-value cut-off of <0.05 and a variance cut-off of >0.005. The sets of differentially expressed probes were then compared to each other. The gene expression array data is available at the NCBI GEO database under the accession designation GSE42265. […]

Pipeline specifications

Software tools GenomeStudio, Qlucore
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Parkinson Disease