Plant Virology Protocols

Kristina Gruden, MaruÅ a Pompe-Novak, Å¡pela Baebler, Hana Krečič-Stres, NataÅ a Toplak, Matjaž Hren, Polona KogovÅ ek, Lisa Gow, Gary D. Foster, Neil Boonham, Maja Ravnikar, MaruŠa Pompe-Novak, špela Baebler, Hana Krečič-Stres, NataŠa Toplak, Matjaž Hren, Polona KogovŠek, Gruden K, Pompe-Novak M, Baebler S, Krecic-Stres H, Toplak N, Hren M, Kogovsek P, Gow L, Foster GD, Boonham N, Ravnikar M

Gary D. Foster, I. Elisabeth Johansen, Yiguo Hong, Peter D. Nagy

Since their conception in the late 1990s, microarray techniques have become a tool of choice for monitoring pangenomic gene expression. Although there are a large number of variations on the basic methodology the general approach remains standard and involves the comparison of a "test" RNA with a "control" RNA; in this case "healthy" and "virus-infected" plants. The protocol itself can be broken down into five main parts: RNA extraction, cDNA synthesis, hybridization, array scanning, and data analysis. The method presented is optimized for use with arrays based on glass slides spotted with cDNA, in this case 15,264 cDNAs from Solanum tuberosum. The labeling technique presented involves two steps: hybridization of cDNA produced using oligo-dT linker primers to the array and hybridization with a DNA dendrimer reagent comprising sequence complementary to the linker sequence bound to a fluorescent dye. We also present the use of the R environment for data analysis, generating statistical support for differential gene expression observed.