Agilent gene expression washing buffers

Gene expression profiling was carried out on the controls (w;tim-Gl4(U)/+) and miR-210 over-expressing flies (w;tim-Gl4(U)/miR-210), sampled at ZT0 and ZT12, using the Drosophila 2.0 custom platform (GPL18767). Four biological replicates were analysed for the controls and miR-210 over-expressing flies, for a total of 8 microarray experiments. Fifty ng of total RNA was labelled with “Low Input Quick Amp Labeling Kit, one color” (Agilent Technologies, CA), following the manufacturer’s instructions. The synthesized cDNA was transcribed into cRNA, labelled with Cy3-dCTP and purified with RNeasy Mini columns (Qiagen, Valecia, CA). The quality of each cRNA sample was verified by the total yield and specificity calculated with NanoDrop ND-1000 spectrophotometer measurements (Nanodrop, Wilmington, DE). Six hundred ng of labelled cRNA were used in each reaction and the hybridization was carried out at 65°C for 17 hours in a hybridization oven-rotator (Agilent Technologies, Palo Alto, CA). The arrays were washed using “Agilent Gene expression washing buffers” and “Stabilization and Drying Solution,” as recommended by the supplier. Slides were scanned on an Agilent microarray scanner (model G2565CA), and the Agilent Feature Extraction software version 10.5.1.1 was used for image analysis. Gene expression data are available in the GEO database using the accession number GSE77245.

Gene expression profiling was carried out on drim2^−/− and drim2^+/−Drosophila larvae using the Drosophila 1.0 custom platform (Agilent Technologies). Total RNA was obtained from the whole body of third instar larvae for each genotype. Four and three biological replicates were analyzed for drim2^−/− and drim2^+/− samples, respectively, for a total of seven microarray experiments. 800 ng of 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 was calculated with NanoDrop ND-1000 spectrophotometer measurements. 1.65 μg of labeled cRNA 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 GSE48012.