normalization data transformation protocol
GPR files were then imported into bioconductor, an open-source software project based on the R programming language. Quantile normalization was accomplished using the normalize.quantiles function. Because the maize oligonucleotide array was designed using an early B73 assembly, all probes were remapped to the latest B73 assembly in Ensembl (AGPv2) using desktop downloaded tBLASTx software and reannotated using BioMart software. The subset of expressed genes was obtained using a method similar to Cassone et al. (2008): probes corresponding to expressed genes were defined as those whose GenPix Pro hybridization signals exceeded 500 for at least three of the six intraline replicates of one maize line and one treatment. Any expressed probes interrogating the same gene were collapsed into a single value by taking the mean normalized probe intensity. The file with normalised data values from both array designs (A-MEXP-1701 and A-MEXP-1702 is Maize_Array_Combined.txt, which can be downloaded from http://www.ebi.ac.uk/arrayexpress/files/E-MTAB-1485/E-MTAB-1485.additional.1.zip
array scanning protocol
Arrays were washed, stained, and scanned using the GeneChip® Fluidics Station 450 and Affymetrix 428 scanner. Raw feature pixel intensity values for each probe were obtained using GenePix Pro 126.96.36.199 software (GenePix).
nucleic acid hybridization to array protocol
labeled cRNA was hybridized to maize oligonucleotide arrays (http://www.maizearray.org) following the recommended protocols
nucleic acid labeling protocol
Total RNA (1µg per sample) was amplified, converted into double-stranded cDNA, and then cRNA amplified and labeled using Cy3 and Cy5 dye randomers with the Amino Allyl MessageAmpTM II aRNA Amplification (Ambion).
Oh28 and Pa405 kernels were inoculated with MDMV followed by 30 degrees C incubation until sample collection at 4 d post-inoculation. Kernels were inoculated by vascular puncture inoculation (VPI). VPI is a highly efficient technique for MDMV infection in maize, with success rates in pre-experiment trials of >90%. Briefly, kernels were soaked in tap water at 30 degrees C for 2.5 h followed by 4 h incubation at room temperature. Kernels were then transferred to damp paper towel. Virus inocula (6 µL per kernel) were prepared using infected plant tissue and 0.01M potassium phosphate buffer (pH 7.0) in a 1:4 ratio (g/mL). Kernels were pierced 8 times per side parallel to the embryo axis using minuten insect pins and an engraver tool. Untreated (mock) controls were handled identically, except VPI was conducted using inocula prepared from healthy plant tissue.
Embryos were excised from the inoculated and control kernels, collected into a 1.5 ml microcentrifuge tube, snap frozen in LN2, and stored at -80 degrees C until RNA isolation. Each sample consisted of 50 excised embryos and was replicated six times per maize line by combining embryos from different inoculation days
Experiments were carried out using two lines of maize: one susceptible to MDMV infection (Oh28) and one resistant to MDMV infection (Pa405). Oh28 was developed in Ohio [(CI.112-1 x Oh90) x (I11.A x I11.B)] and released in 1943. Pa405 was developed in Pennsylvania [NY3 x Pa54] and released in 1972. Both maize lines were stored as seeds at the Ohio Agricultural Research and Development Center (OARDC) at room temperature until experimentation. Maize dwarf mosaic virus-OH2 (MDMV-OH2, GenBank accession number JQ403609) was originally collected in Southern Ohio ca. 1970 by D. Gordon and associated and was maintained in ‘Spirit’ maize by mechanical rub inoculation.
nucleic acid extraction protocol
Total RNA was isolated and purified from pools of 50 kernels using a standard guanidine thiocyanate procedure. Extracted RNA was purified using the RNeasy MinElute Cleanup Kit (QIAGEN). RNA quality and quantity were assessed using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies) at wavelengths of 230 nm, 260 nm, and 280 nm. The integrity of total RNA was further verified by running 200 ng samples on 1.5% agarose gels. Total RNA (5 µg per sample) was treated with DNase 1 (Invitrogen) to remove any contaminating DNA.