Mircury array power labeling kit

Total RNA of plasma samples was isolated using Trizol LS (Invitrogen, Canada) and purified with an RNeasy mini kit (Qiagen, Germany), and the quantity of RNA was determined using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, US). A miRCURY™ Array Power Labeling kit (Exiqon, Denmark) was used for microRNA labeling, and then Hy3™-labeled samples were hybridized on a miRCURY™ LNA Array (Exiqon). Chips were scanned using an Axon GenePix 4000B microarray scanner (Axon Instruments, CA, US) and the images were imported into GenePix Pro 6.0 software (Axon Instruments, CA, US) for grid alignment and data extraction. The replicated microRNAs were averaged and those with intensities ≥ 30 in all samples were selected for normalization. Differentially expressed microRNAs were identified by a volcano plot and a heat map (MEV ver. 4.8, TIGR).

RNA of sufficient quality from eight group samples (n = 3, total 24 samples) was submitted to KangChen-Biotech (Shanghai, China). RNA groups above mentioned by applying the miRCURY locked nucleic acid (LNA) microarray platform (Exiqon, Denmark). All procedures were carried out according to manufacturer’s protocol. Briefly, 1 μg total RNA was labeled with the Hy3™ or Hy5™ fluorophores, using miRCURY™ Array Power Labeling kit (Exiqon, Denmark). After stopping the labeling procedure, briefly spin the reaction and leave it at 4 °C. The two samples from the Hy3™ and Hy5™ labeling reactions are combined on ice. The samples were hybridized on a hybridization station using miRCURY™ LNA miRNA array (v.18.0) containing Tm-normalized probes for 847 human miRNAs. Microarrays with labeled samples were hybridized at 56 °C for overnight using a heat-shrunk hybridization bag and washed using miRCURY Array Wash buffer kit (Exiqon, Denmark). After hybridization, the chip slides were washed, dried, and scanned immediately. Each miRNA spot was replicated for four times on the same slide and two microarray chips have been used for each group.

miRNA was labeled using the miRCURY™ Array Power Labeling kit (cat no. 208032-A; Exiqon, Inc., Woburn, MA, USA) in accordance with the manufacturer’s instructions. Briefly, 4 μl calf intestine phosphatase (CIP) reaction solution (1 μl total RNA, 0.5 μl CIP buffer, 0.5 μl CIP and 2 μl ddH₂O) was incubated at 37°C for 30 min and then at 95°C for 1 min to terminate the reaction, and immediately placed in an ice bath for 10 min. Following mild centrifugation (Sigma 4K 15CR; 200 × g), 3 μl labeling buffer, 1.5 μl fluorescent labels (Hy3™ for the stress group or Hy5™ for the control), 2 μl DMSO and 2 μl labeling enzyme were sequentially added in an ice bath. The system was incubated at 16°C for 1 h and subsequently at 65°C for 15 min in order to terminate the labeling reaction, and stored at 4°C following mild centrifugation.

The preparation of RNA samples and microarray analysis were performed commercially
by RiboBio Co. Ltd (Guangzhou, China). In brief, total RNA of LECs treatment with
or without TGFβ2 was isolated using TRIzol (Invitrogen, Carlsbad,
CA, USA) and miRNeasy mini kit (QIAGEN, Hilden, Germany) according to the
manufacturer’s instructions, which efficiently recovers all RNA species,
including miRNAs. RNA quality and quantity were measured by nanodrop
spectrophotometer (ND-1000, Nanodrop Technologies, Wilmington, DE, USA), and RNA
integrity was determined by gel electrophoresis. The isolated miRNAs were then
labeled with Hy3/Hy5 using the miRCURY Array Power Labeling kit (Exiqon,
Vedbaek, Denmark) and hybridized on a miRCURY LNA miRNA Array (v.18.0, Exiqon)
according to array manual. Following hybridization, the slides were achieved,
washed several times using wash buffer kit (Exiqon) and finally dried by
centrifugation for 5 min at 400 r.p.m. Then the slides were scanned using
the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA,
USA). The scanned images were then imported into GenePix Pro 6.0 software (Axon
Instruments) for grid alignment and data extraction. Bioinformatics analysis and
visualization of microarray data were performed with MEV software (v4.6, TIGR).
The microarray assays were repeated three times each group.

Microarray analysis was performed using Agilent Human miRNA (8*15K) V14.0 arrays of Ribobio (Guangzhou, China), as described previously [15 (link)]. miRs expression profiling was detected using miR microarray assays (n = 3 in each group). miRNeasy Mini Kit (Qiagen, Inc., Valencia, CA, U.S.A.) was used to extract total RNA, according to the manufacturer’s protocol. miRs were labeled with Hy3 or Hy5 fluorescence using the miRCURY™ Array Power Labeling Kit (Exiqon) to obtain the fluorescent probe that can be hybridized with the chip. The labeled probe was hybridized with the miRCURY™ chip under the standard condition using the MAUI hybridization system. Agilent scanner and the Feature Extraction 10.7.1.1 software (Agilent Technologies) were used to obtain the microarray raw data. Microarray results were analyzed using the GeneSpring GX 12.5 software (Agilent Technologies). Differentially expressed miRs were selected out according to |Log2fold change| ≥ 1, P < 0.05 and false discovery rate < 0.05. The hierarchical clustering analysis was performed using MeV software (version 4.2.6).

Six mice were randomly divided into normal control group (NC) and CFA group. One day after CFA treatment, TGs from the unilateral surface were removed quickly and immediately frozen in liquid nitrogen for miRs assay. Total RNA in TGs was extracted by TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). miRs were isolated from total RNA using the miRNA isolation kit (Invitrogen). Denaturing agarose gel electrophoresis was performed using 1% formaldehyde electrophoresis reagent. miRs were labeled with Hy3 or Hy5 fluorescence using the miRCURY™ Array Power Labeling Kit (Exiqon, Denmark) to obtain the fluorescent probe that can be hybridized with the chip. The labeled probe was hybridized with the miRCURY™ chip under the standard condition using the MAUI hybridization system. The fluorescence intensity of the chip was scanned with the Agilent chip scanner and analyzed using Agilent feature extraction software (version 12). The differentially expressed miRs were screened based on the fold change ≥2, P<0.05 and FDR<0.05. Finally, the differentially expressed miRs in TGs were displayed by hierarchical clustering analysis between the two groups.

The RNA samples from B cells isolated from PBC patients or healthy control were labeled using a miRCURY^™ Array Power Labeling kit (Exiqon, Vedbaek, Denmark). Next, the labeled RNA was hybridized on a miRCURY^™ LNA Array (v.18.0, Exiqon). Array slides were scanned using an Axon GenePix 4000B Microarray Scanner (Axon Instruments, Foster, USA) Data were extracted from scanned images using GenePix Pro 6.0 (Axon Instruments). All the microarray analysis was performed by the Exiqon miRNA Expression Profiling service (Kangchen Bio-tech, China).