Gene Expression Analysis of In Vivo Fluorescent Cells

Abstract

BackgroundThe analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters.Methodology/Principal FindingsWe developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of ∼2∶1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR).Conclusions/SignificanceOur technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.

Cell type-specific transcriptome analysis in the early Arabidopsis thaliana embryo

“…The sorted positive nuclei were collected in RNA extraction buffer [10 mM Tris-HCl ( pH 7.9), 50 mM EDTA ( pH 7.9), 0.2 M NaCl, 0.5% SDS, 0.5 mg/ml RNase inhibitor (Fermentas), 600 µg/ml proteinase K] (Khodosevich et al, 2007). The buffer containing the GFP-positive nuclei was incubated at 55°C with vigorous shaking for 10-15 min.…”

Epigenetic regulation of neurogenesis in the adult hippocampus

“…Epigenome profiling of neurogenesis in adult hippocampus is technically challenging as it requires the analysis of many distinct developmental stages. Methods such as the retroviral labeling of newborn neurons by stereotactic injections into the adult hippocampus (Jagasia et al, 2009) and laser microdissection of labeled cells (Khodosevich et al, 2007) could be used to specifically select the neural stem cells and their progeny at distinct developmental stages. Yet, the major bottleneck is the development of low/single cell-based high-throughput sequencing for epigenome profiling of stem cells and their neuronal progeny in the adult brain.…”

Differential Gene Expression in Gonadotropin-Releasing Hormone Neurons of Male and Metestrous Female Mice

“…Brain fixation, preparation of sections for the subsequent LCM and RNA isolation were performed as reported elsewhere [46] with minor modifications. Briefly, male (n = 6) and metestrous female (n = 6) mice were deeply anesthetized with ketamine/xylazine (100 and 10 mg/kg body weight, respectively) and perfused transcardially at a flow rate of 8 ml/min with 80 ml 0.5% paraformaldehyde in DEPC-treated PBS (pH 7.4), followed by 20% sucrose in DEPC-PBS.…”