Laser Capture Microdissection and Real-Time PCR for Measuring mRNA in Giant Cells Induced by Meloidogyne javanica
Keywords:gene expression, giant cells, hxk1, lah4, laser capture microdissection, meloidogyne javanica, mrna, rb7, root-knot nematodes, real-time pcr, reverse transcription
AbstractThe techniques of laser capture microdissection and quantitative RT-PCR were investigated as methods for measuring mRNA in giant cells induced by Meloidogyne javanica. Laser capture microdissection allowed precise sampling of giant cells at 1 to 3 weeks after inoculation. The expression of three genes (a water channel protein gene Rb7, a plasma membrane H[sup+]-ATPase (LHA4), and a hexose kinase (HXK1) was measured based on mRNA extracted from tissue samples and quantitated using reversetranscription real-time PCR. These genes were chosen arbitrarily to represent different aspects of primary metabolism. The amount of HXK1 mRNA in giant cells was not different from that in root meristem or cortical cells when compared on the basis of number of molecules per unit tissue volume, and was similar at all sample times. Amount of mRNA for LHA4 and Rb7 was much greater in giant cells than in cortical cells, but only Rb7 was also greater in giant cells than in root meristem cells. Numbers of mRNA molecules of LHA4 increased linearly in giant cells from 1 to 3 weeks after inoculation, whereas the amount of Rb7 mRNA was similar at 1 and 2 weeks after inoculation but increased at 3 weeks after inoculation. The amount of mRNA for these two genes was similar at all sample times in cortical and root-tip cells. Apparent up regulation of some genes in giant cells may be due primarily to the increased number of copies of the gene in giant cells, whereas for other genes up regulation may also involve increased transcription of the increased number of copies of the gene.
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