Total RNA was isolated using TRIzol (Invitrogen) and purified with RNeasy mini kit (QIAGEN) according to manufacturer’s instructions. RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel electrophoresis.
Label
Cy3
Label protocol
RNA labeling and array hybridization was according to Exiqon's manual.After quality control, the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling by following steps: a, 1μLRNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C. b, The Reaction was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3TM), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture.The labeling reaction was incubated for 1 h at 16°C. c,Terminated by incubation for 15 min at 65°C.
Hybridization protocol
After stopping the labeling procedure, the Hy3™-labeled samples were hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon) according to array manual.a,The total 25 μL mixture from Hy3™-labeled samples with 25 μL hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min. b, Then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization Systems (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA). c, Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon)
Scan protocol
Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).
Data processing
Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities>=30 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. Discriminant miRNAs and differences between groups were analyzed using Bayes moderated t test (limma) with Benjamini Hochberg false discovery rate (FDR) at P < 0.05. A two-fold cut-off difference was applied to select the up- and down-regulated miRNAs.
Submission date
Jan 28, 2016
Last update date
Feb 22, 2018
Contact name
Shizhi Wang
Organization name
Southeast University
Department
Key Laboratory of Environmental Medicine Engineering, Ministry of Education
