P-CAGE-26808 - P-CAGE-26808
Total RNA from sperm cells, pollen and seedlings was extracted using the RNeasy Micro Kit (Qiagen, Germany). RNA integrity was assessed using an Agilent 2100 Bioanalyser with a RNA 6000 Nano Assay (Agilent Technologies, Palo Alto, CA). RNA was processed for use on Affymetrix Arabidopsis ATH1 Genome Arrays (Santa Clara, CA, USA) according to the manufacturer’s Two-Cycle Target Labeling Assay. Briefly, 16 ng of total RNA containing spiked-in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix) was used in a reverse transcription reaction (Two-Cycle DNA synthesis kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an in vitro transcription (IVT) reaction to generate cRNA (MEGAscript T7 kit; Ambion, Austin, TX). The cRNA obtained was used for a second round of cDNA and cRNA synthesis, resulting in biotinylated cRNA (GeneChip Expression 3’-Amplification Reagents for IVT-Labeling; Affymetrix). The size distribution of the cRNA and fragmented cRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay. Fifteen micrograms of fragmented cRNA was used in a 300-µl hybridization containing added hybridization controls. 200 µl of the mixture was hybridized on arrays for 16 h at 45°C. Standard post-hybridization washes and double-stain protocols (FS450_0004) were used on an Affymetrix GeneChip Fluidics Station 450, in conjunction with the GeneChip Hybridization Wash and Stain kit (Affymetrix). Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.
Amount of nucleic acid labeled, Amplification, Used Label