Using array hybridization to monitor gene expression at the single cell level.
Advances in high-throughput genome sequencing demand the development of more efficient ways of examining gene expression at a cellular level. During recent years, polymerase chain reaction (PCR)-based methods have been developed that allow the amplification of mRNA from small amounts of material, even from single animal cells. In parallel, several analytical tools permit a global monitoring of gene expression. To date, high throughput analysis methods have not been accessible for single plant cell samples. In the protocol described here, cDNA array hybridization (expression profiling) and an amplification strategy using reverse transcriptase PCR are merged with high spatial resolution sampling from undamaged plant tissue. This protocol gives us a new tool to examine tissue-specific gene expression patterns on a comprehensive scale. To demonstrate the usefulness of this tool, gene expression patterns in samples from Arabidopsis thaliana L. cv. C24 leaf epidermal and mesophyll cells were measured; several differentially expressed genes were identified when single cell samples were compared. The protocol described has the potential of increasing the efficiency of tissue-specific expression analysis by combining high-throughput profiling with straightforward sampling and amplification procedures.
Brandt
Stephan
S
Max Planck Institut für Molekulare Pflanzenphysiologie, Department Willmitzer, Am Mühlenberg 1, D-14476 Golm, Germany.
Kloska
Sebastian
S
Altmann
Thomas
T
Kehr
Julia
J
D017360:Arabidopsis / Q000166:cytology / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D015870:Gene Expression; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers