Computational protocol: Multipotent Caudal Neural Progenitors Derived from Human Pluripotent Stem Cells That Give Rise to Lineages of the Central and Peripheral Nervous System

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Protocol publication

[…] hESC, day 4 and day 11 time points of the various differentiation conditions were collected by mechanical isolation, all conditions were analyzed from three independent biological replicates. Total RNA was isolated from cells using the RNeasy Mini RNA Extraction Kit (Qiagen) and contaminant genomic DNA removed with DNA‐free DNaseI reagents (Ambion, Life Technologies). Primer sequences were designed using the primer3 tool (http://frodo.wi.mit.edu/primer3/). For quantitative reverse transcription and polymerase chain reaction (Q‐PCR), oligo‐dT primed cDNA was synthesized from 130 ng DNaseI‐cleaned total RNA using Murine Moloney Leukemia Virus reverse transcriptase (Promega, Alexandria, Australia, www.promega.com). Q‐RTPCR was performed on an ABI Prism 7500 Fast Real‐Time PCR System (Applied Biosystems, Life Technologies) using Go‐Taq SYBR green master mix (Promega). Relative gene expression values (mRNA fold change) were obtained by normalization to the internal reference genes RPL32 using the −2ΔΔCt method, where −2ΔΔCt = ΔCt sample − ΔCt calibrator as described . Hierarchical clustering and heatmap analysis of Q‐PCR data were done using R‐script and gplots packages. […]

Pipeline specifications

Software tools Primer3, gplots
Application qPCR
Organisms Homo sapiens