title: Quantitative large scale gene expression profiling from human stem cell culture micro samples using multiplex pre-amplification.
abstract: Transcriptional profiling is a powerful tool to study biological mechanisms during stem cell differentiation and reprogramming. Genome-wide methods like microarrays or next generation sequencing are expensive, time consuming, and require special equipment and bioinformatics expertise. Quantitative RT-PCR remains one of today's most widely accepted and used methods for analyzing gene expression in biological samples. However, limitations in the amount of starting materials often hinder the quantity and quality of information that could be obtained from a given sample. Here, we present a fast 4-step workflow allowing direct, column-free RNA isolation from limited human pluripotent stem cell (hPSC) cultures that is directly compatible with subsequent reverse transcription, target specific multiplex pre-amplification, and standard SYBR-Green quantitative PCR (qPCR) analysis. The workflow delivers excellent correlations in normalized gene-expression data obtained from different samples of hPSCs over a wide range of cell numbers (500-50,000 cells). We demonstrate accurate and unbiased target gene quantification in limiting stem cell cultures which allows for monitoring embryoid body differentiation and induced pluripotent stem cell (iPSC) reprogramming. This method highlights a rapid and cost effective screening process, allowing reduction of culture formats and increase of processing throughputs for various stem cell applications.
authors.affiliation: a Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital , Toronto , Canada .
authors.forename: Mark
authors.initials: M
authors.lastname: Kibschull
authors.affiliation: a Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital , Toronto , Canada .|b Departments of OBS/GYN, Physiology, and Medicine , University of Toronto , Toronto , Canada .|c Fraser Mustard Institute for Human Development, University of Toronto , Toronto , Canada .
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lye
authors.affiliation: d Gene Expression Division , Life Science Group, Bio-Rad Laboratories , Hercules , California , USA , and.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Okino
authors.affiliation: e Gene Expression Division , Life Science Group, Bio-Rad Laboratories , Mississauga , Ontario , Canada.
authors.forename: Haya
authors.initials: H
authors.lastname: Sarras
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D003362:Cost-Benefit Analysis; D058732:Embryoid Bodies; D005784:Gene Amplification / Q000235:genetics; D020869:Gene Expression Profiling / Q000191:economics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057026:Induced Pluripotent Stem Cells; D046228:Microarray Analysis; D016133:Polymerase Chain Reaction; D013234:Stem Cells / Q000378:metabolism*; D057188:Workflow
keywords: Differentiation; embryoid body; pre-amplification; real-time PCR; reprogramming; stem cell