One color microarray based gene expression protocol

Gene expression profiling was carried out on controls and Surf1 Act-Gal4 KD first instar Drosophila larvae using the Drosophila 1.0 custom platform (Agilent Technologies). Total RNA was obtained from the whole body of 1st instar larvae for each genotype. Four biological replicates were analyzed for controls and Surf1 KD samples, respectively for a total of 8 microarray experiments. 800 ng total RNA was labeled with “Agilent One-Color Microarray-Based Gene Expression protocol” according to the manufacturer's instructions. The synthesized cDNA was transcribed into cRNA and labeled with Cy3-dCTP. Labeled cRNA was purified with RNeasy Mini columns (Qiagen). The quality of each cRNA sample was verified by total yield and specificity calculated with NanoDrop ND-1000 spectrophotometer measurements. Labeled cRNA (1.65 μg) was used in each reaction, and hybridization was carried out at 65 °C for 17 h in a hybridization oven rotator (Agilent). The arrays were washed using Agilent Gene expression washing buffers and Stabilization and Drying Solution, as suggested by the supplier. Slides were scanned on an Agilent microarray scanner (model G2565CA), and Agilent Feature Extraction software version 10.5.1.1 was used for image analysis. Gene expression data are available in the GEO database with the accession number GSE58301.

Gene expression profiling was carried out in an ERMS cell line (RH36) transfected with pre-miR-27a or with a negative pre-control by using the ‘‘Whole Human Genome Oligo Microarray” (Agilent), consisting of 41,000 (60-mer) oligonucleotide probes, which span conserved exons across the transcripts of the targeted full-length genes. Each slide contains four individual microarrays, every one having about 44,000 features.
Eight hundred ng of total RNA were labeled with ‘‘Agilent One-Color Microarray-Based Gene Expression protocol” in accordance with the manufacturer’s instructions. Using 1.65μg of labeled cRNA to prepare the hybridization samples, the hybridization was carried out at 65°C for 17 hours in a hybridization oven rotator (Agilent). The arrays were washed with Agilent Gene expression wash buffers and Stabilization and Drying Solution, as suggest by the supplier. Slides were scanned on an Agilent microarray scanner (model G2565CA), and Agilent Feature Extraction software version 10.5.1.1 was used for image analysis. Raw data are available on the GEO website using accession number GSE52678.

Gene expression profiling was carried out in krill sampled at different times throughout the 24 hours (day times: 6:00, 9:00, 12:00, 15:00, 18:00, 21:00, 24:00, 3:00 and 6:00) in LD and DD conditions with the Krill 2.0 custom platform (Agilent). In every entrainment condition, for each time point, a total of three biological replicates were considered, for a grand total of 54 microarrays. As described in De Pittà et al.^{14 (link)}, equal amounts of total RNA extracted from three different specimens were mixed to prepare each biological replicate. 800 ng of total RNA were labelled with “Agilent One-Color Microarray-Based Gene Expression protocol”. Slides were scanned on an Agilent microarray scanner (model G2565CA) and Agilent Feature Extraction software version 10.5.1.1 was used for image analysis. Gene expression data are available in GEO database with the accession number: GSE94757.