Cowpea genotype IT93K-503-1. Tissue was isolated 14 days after planting. Tisuue type young trifolilate leaves. Plants were grown under normal irrigation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using TRIzol (Gibco BRL Life Technologies, Rockville, MD) reagent. RNA was further purified using an RNeasy spin column (Qiagen, Chatsworth, CA) and an on-column DNase treatment. RNA integrity was assessed prior to target preparation using RNA Lab-On-A-Chip (Caliper Technologies Corp., Mountain View, CA) evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
Single-stranded, then double-stranded cDNA was synthesized from the poly(A)+ mRNA present in the isolated total RNA (10 µg total RNA starting material each sample reaction) using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen Corp., Carlsbad, CA ) and poly (T)-nucleotide primers that contained a sequence recognized by T7 RNA polymerase. A portion of the resulting ds cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction (IVT), using the BioArray High-Yield RNA Transcript Labeling Kit (T7) (Enzo Diagnostics, Inc., Farmingdale, NY).
Hybridization protocol
Biotin labelled cRNA was hybridized to an Affymetrix soybean genome array as recommended by Affymetrix (Affymetrix GeneChip Expression Analysis Technical Manual; Affymetrix Inc., Santa Clara, CA) at the Institute for Integrative Genome Biology Microarray Facility at the University of California, Riverside (http://www.genomics.ucr.edu).
Scan protocol
The GeneChip arrays were washed and then stained (SAPE, streptavidinphycoerythrin)on an Affymetrix Fluidics Station 400, followed by scanning on a Hewlett-Packard GeneArray scanner.
Description
Cowpea_503-1_UnStressed_Replicate2
Data processing
The hybridization data were scanned for visible defects and then extracted using default settings and tabulated as CEL files using Affymetrix GeneChip Operating Software (GCOS 1.2). A global scaling factor of 500, a normalization value of 1, and default parameter settings were used for the soybean genome array.
