Abstract
BACKGROUND: The 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 FINDINGS: We 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 approximately 2ratio1. 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/SIGNIFICANCE: Our 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.
| Original language | English |
|---|---|
| Journal | PLOS ONE |
| Volume | 2 |
| Issue number | 11 |
| Pages (from-to) | e1151 |
| ISSN | 1932-6203 |
| DOIs | |
| Publication status | Published - 7 Nov 2007 |
| Externally published | Yes |
Keywords
- Animals
- Brain/metabolism
- Fluorescence
- Gene Expression Profiling
- Green Fluorescent Proteins/genetics
- Mice
- Mice, Transgenic
- Oligonucleotide Array Sequence Analysis
- Receptors, Serotonin, 5-HT3/genetics
- Reverse Transcriptase Polymerase Chain Reaction