Minisymposium 21: Emerging technologies
Abs #
42001: Solid Phase Gene Extraction - repeated sampling of mRNA from single cells
The need for repeated but independent extractions of mRNA from single cells and plant tissues prompted the development of Solid Phase Gene Extraction (SPGE). In a chemical reaction, oligo dT nucleotides are covalently bonded to the surface of borosilicate needles with 0.5 μm diameter. The coated glass needles are inserted into a single living cell or tissue where the oligo dT18 nucleotides hybridize with the poly A+ mRNA. After withdrawal, the needle can be used directly for RT-PCR. Because of the small probe size, no cytoplasm is lost, the cell remains healthy and repeated sampling of the same cell is possible. After reverse transcription, the cDNA is covalently linked to the needle, which then can serve as a cDNA library.
SPGE of Chara rhizoids showed fluctuations of type and quantity of mRNA in specific areas of the cytoplasm. Sampling of internodal cells revealed time-dependent gene expression as a function of light/dark intervals. Despite extensive cytoplasmic streaming, samples taken in the vicinity of the nucleus were more variable than samples from the ends of the cell. In Arabidopsis, we isolated cDNA species from root tips, shoots and leaves and determined their sequences. Gene-specific primers allowed the monitoring of gene expression over time. After a short recovery period SPGE-sampled roots and shoots showed normal growth rates and graviresponse. These data indicate that SPGE is a powerful approach to sample and study gene expression in single cells and in tissues of higher plants with high spatial and temporal resolution without significantly affecting regular growth responses. So far, SPGE is the only technology that permits repeated non-destructive sampling of genetic material. Supported by NASA: NAG 2-1423
