One colour rna spike in kit

For transcript profiling, RNA was purified using RNeasy Mini Spin Columns (Qiagen) following the manufacturer’s protocol. Quality and quantity was measured with an Agilent RNA 6000 Nano Chip on an Agilent 2100 BioAnalyzer (version B.02.03 BSI307) following by Agilent RNA 6000 Nano Assay Protocol2. Sample labelling and preparation for microarray hybridization were performed as described in the one-colour microarray-based gene expression analysis protocol—provided by Agilent—including the one-colour RNA spike-in kit (Agilent Technologies). Cy3-labelled samples were fragmented and loaded on the array (Agilent Arabidopsis V4 – Design number 21169), hybridized overnight (17h/65 °C), and washed. Slides were scanned on the Agilent Microarray Scanner with extended dynamic range at high resolution.

The RNA extracted from the leaf samples was prepared for microarray hybridisation at the Genomic Service of the IBMCP Institute (Instituto de Biología Molecular y Celular de Plantas) in Valencia (Spain) by Agilent technologies. cDNA synthesis and labelling on Agilent Tomato microarrays were carried out using the Agilent One Colour RNA Spike-in Kit and the Agilent Low Input Quick Amp Labeling Kit. Microarray hybridisation and washing were next performed with the Agilent Gene Expression Hybridization kit and Gene Expression Wash Buffers. Agilent microarray 4*44 k (Agilent G2519F) was selected for hybridisation (reference AMADID 22270 Tomato). Microarray scanning was done with a GenePix 4000B (Axon Molecular Devices, Sunnyvale, USA) and data were extracted by the Agilent Feature Extraction software, version 9.5.1.