National Facility for DNA Microarrays - University of Aveiro
Manufacture protocol
The spotted oligo library was the NCodeTM multi-species miRNA probe set V2 from Invitrogen. This library contains 1164 34-44mer unmodified DNA oligonucleotides targeting human, rat and mouse, Drosophila, C.elegans and zebrafish mature miRNA sequences from miRBase Sequence Database version 9.0. In addition, the mirVana miRNA Probe Set includes 24 probes targeting an exclusive set of newly identified zebrafish miRNAs, one positive control and several mismatch controls for screening 1- and 2-base sequence mismatches. The library is provided in 384-well plates in lyophilized form. LIQUID HANDLING: The goal was 20 uM oligos in 150 mM phosfate buffer in 384-well plates (Genetix X-6004) ready for robotic spotting. 1. Lyophilized oligos (500 pmol in 384-well plates) were resuspended in 150 mM phosphate buffer to a concentration of 20 uM using a liquid handling station (Sciclone LH 3000, Caliper). 2. Shaking overnight at 4 ºC, at 100 rpm. The 384-well plate was stored at -80 ºC ready for spotting. 3. The remaining stock in 384-well plates was air dried at room temperature after spotting and stored at -80 ºC. When necessary, milliQ water was used to resuspend the oligos accounting for lost volume. SPOTTING: 1. The 34-44mer probes were transfered in quadruplicate from the 384-well plates and printed on Nexterion E slides (SCHOTT) with a BioRobotics MicroGrid Compact II spotter (Genomic Solutions) in an environment controlled room. Humidity was 60% ± 2%. Temperature was 21 ºC in the spotter. Quill Pins type MicroSpot2500 (BioRobotics) were used for spotting. 2. For probe fixation, the printed slides were incubated in a humidity chamber at room temperature, overnight. 3. Slides were stored in a desiccator-cabinet (Nalgene, with blue Silica (Sigma)) ready to use (continue with our hybridization protocol).
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