array scanning protocol
The miRCURY LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies). The microarray slides were scanned and stored in an ozone-free environment in order to prevent potential bleaching of the fluorescent dyes. Image analysis was carried out using the ImaGene 8.0 software (BioDiscovery). (Parameters: Scanning hardware = G2565BA DNA microarray scanner [Agilent], Scanning software = AIDA [Raytest GmbH])
The hybridization was performed according to the miRCURY LNA array manual using a Tecan HS4800 hybridization station (Tecan). Each Hy3(tm)-labeled sample was mixed with the Hy5(tm)-labeled reference RNA sample and then hybridized on the miRCURY LNA(tm) miRNA Arrays (v.11.0) following the common reference design. (Parameters: Chamber type = OTHER: Tecan HS4800 hybridization station, Quantity of label target used = 1, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume unit = Nano litre, temperature = 56)
nucleic acid labeling protocol
Total RNA samples (1 ug) and reference RNA samples were labeled with Hy3(tm) and Hy5(tm) fluorescent labels, respectively, using the miRCURY(tm) LNA Array power labeling kit (Exiqon Vedbaek), following the procedure described by the manufacturer. (Parameters: Amount of nucleic acid labeled = 1, Amplification = none, Mass unit = Micro gram)
nucleic acid extraction protocol
Add 1 ml Trizol to tissue sample and homogenize with machine in Corex tube. Incubate at RT for 5 min and freeze in –80. Continue with RNA extraction using ultracentrifuge: Rotor ss-34 code 05. After the removal of the RNA phase for recovery of small RNAs: X2 volume of isopropyl alcohol and placing the solution at -80C for two hours before centrifuge. (Parameters: Extracted product = total_RNA, Amplification = none)
To create a common reference pool, Exiqon pooled all samples per experiment for the use of Hy5 signals. The common reference was a pool of RNA composed of ten samples: four control samples, three samples of 24 hrs after E2 treatment and three samples of 48 hrs after E2 treatment.
Male zebrafish were exposed to 17beta-estradiol (E2) (Sigma) by immersion. The concentration used was 5 ug/L (18 nM). Ten fish were kept in each aquarium (5L in volume) under static conditions. E2 (dissolved in 50 % Ethanol) was added and tissue samples were collected at each time point (0, 24, 48 hrs) after treatment. Water was changed and E2 was refreshed once a day during the incubation period. Only Ethanol was added to control (0 hrs) fish. Samples were frozen instantly in liquid nitrogen and stored at -80C.