title: miRNAChip_human_V2
status: Public on Aug 01 2012
contact.city: Aveiro
contact.first: Patrícia
contact.middle: Matos
contact.last: Pereira
contact.email: pereirap@ua.pt
contact.address: Campus Universitário de Santiago
contact.department: Biology
contact.country: Portugal
contact.institute: University of Aveiro
organism: Homo sapiens
manufacture_protocol: The arrays contained a total of 1175 probes spotted in quadruplicate. Each array was printed with optimized probe sequences targeting all of the 703 known human mature miRNAs deposited in the Sanger miRBase Sequence Database, Release 10.0 (http://microrna.sanger.ac.uk), as well as 393 novel human miRNAs discovered through deep sequencing and validated by qRT-PCR and array profiling (NCode™ Human miRNA microarray probe set V3 from Invitrogen). The probe set also includes mismatch controls for screening 1- and 2-base sequence mismatches for monitoring hybridization specificity, as well as the NCode™ Positive Control for labeling quality control and easier spot finding. These unmodified oligonucleotides, which are 34–44 bases in length, were dissolved in 150 mM phosphate buffer pH 8.5 at a concentration of 20 µM and printed onto Nexterion E slides (SCHOTT) using a MicroGrid II compact spotter. Printed slides were further processed according to manufacturer's recommendations. 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 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).