Cip enzyme

After extraction, RNA samples underwent quality control and miRNA was labeled using miRCURYTM Array Power Labeling kit (Cat #208032-A, Exiqon). The detailed steps are described as follows:One microgram RNA sample was added to 2 μL water, and then 1 μL CIP buffer and CIP enzyme (Exiqon) were added to the RNA sample solution, and the mixture solution was incubated at 37°C for 30 min. The reaction in the sample solution was terminated at 95°C for 5 min, and then 3 μL labeling buffer, 1.5μL fluorescent label (Hy3^™), 2.0 μL DMSO, and 2.0 μL labeling enzyme were added and the solution was incubated at 16°C for 1 h. The reaction in the samples was terminated at 65°C for 15 min.
After labeling, the sample was hybridized with miRCURYTM LNA Array (v.18.0) (Exiqon), and the subsequent procedures were conducted according to Exiqon's experimental methods. The hybridization system used was the Nimblegen system (Nimblegen Systems Inc., Addison, WI, USA). The microarray chip was rinsed with Wash buffer kit (Exiqon) after hybridization.

RNA labeling and array hybridization were performed according to the Exiqon manual. The miRCURY™ Hy3™/Hy5™ Power Labeling Kit (Exiqon, Vedbaek, Denmark) was used for miRNA labeling after quality control. RNA (1 µg in 2 µL of water) was combined with 1 µL of calf intestine phosphatase (CIP) buffer and CIP enzyme (Exiqon), and incubated for 30 min at 37 °C. The reaction was terminated by incubation for 5 min at 95 °C. Then, 3 µL of labeling buffer, 1.5 µL of fluorescent label (Hy3TM), 2 µL of DMSO, and 2 µL of labeling enzyme were added to the mixture. The labeling reaction was incubated for 1 h at 16 °C, and then terminated by incubation for 15 min at 65 °C. After stopping the labeling procedure, the Hy3™-labeled samples were hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon) according to the array manual. The Hy3™-labeled samples (25 µL), mixed with 25 µL hybridization buffer, were first denatured for 2 min at 95 °C, incubated on ice for 2 min, and then hybridized to the microarray for 20 h at 56 °C in a 12-Bay Hybridization System (Hybridization System-Nimblegen Systems, Inc., Madison, WI, USA). Following hybridization, the slides were washed several times with a wash buffer kit (Exiqon) and then scanned using an Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA, USA).