A simple method for obtaining cell-specific cDNA from small numbers of growing root-hair cells in Arabidopsis thaliana
Journal of Experimental Botany, 2003, 54(386): 1373-1378.
A simple and rapid method for cloning specific cDNAs from mRNA populations derived solely from small numbers of root-hair cells is described here. To identify genes expressed during the earliest visible stage of root-hair cell development, cell contents were aspirated from small numbers of Arabidopsis root-hair cells at or just before this stage. This material was used to make reusable solid-phase oligo-dT-primed cDNA libraries. To demonstrate that the libraries contained high quality longer cDNAs, a fragment located 2.7 kb from the 3' end of the cDNA of the single copy root-hair expressed gene RHD3 was cloned using a nested PCR strategy. This technique was also used to obtain novel gene expression information by cloning the full-length 0.85 kb cDNA of the Rop2 GTPase from this library. This approach offers a means of cloning larger cDNAs directly from small numbers of growing root-hair cells and, potentially, other epidermal cell types.
cell aspiration; complementary DNA; messenger RNA; nested PCR; root hairs