Solid Phase Gene Extraction (SPGE)

Although much information can be gained from direct measurements of physical parameters, the ultimate control in living systems depends on information processing.In addition to using artificial statoliths to investigate force- and relocation effects on gravitropism (in Chara) it is important to investigate gene expression as a response to physiological stimulation. We are in the process of developing a novel mRNA extraction procedure that relies on glass needles (= solid phase) that is poly-T coated. This procedure can extract information of gene activation in different regions of a cell, not just the total mRNA that is typically obtained by processing entire cells. The fast and reliable in situ sampling shows remarkable consistency in the PCR-amplified mRNA signature. We have observed diurnal and spatial differences in single Chara internodal cells.

Chara internodal cell impaled with a SPGE probe. The same cell can be sampled up to 16 times.

Illustration of the potential and sensitivity of SPGE: A Chara internodal cell was probed at two different positions followed by RT-PCR (amplification with a dT18 primer and 4 random primer). The samples (groups of 4 lanes, left to right) were taken from the periphery of the cell. The first and third group were taken one h after the light in the growth chamber was turned on, samples two and four were taken one h after dark. The similarity between the samples indicates a diurnal pattern and that SPGE is a meaningful approach for the spatial and temporal analysis of gene expression in single cells.