Human mirna microarray kit

Calcific aortic valves and non‐calcific valves from three CAVD patients were sent to carry on the miRNA microarray assay. Total RNA was extracted from tissues using the miRNAeasy Mini Kit (Qiagen GmbH). The miRNA microarray assay was performed by a service provider (LC Sciences). Total RNA (100 ng) was labelled with miRNA Complete Labeling and Hyb Kit (Agilent, USA) and hybridized on the Human miRNA Microarray Kit (Release 16.0, Agilent), which contains 60 000 probes for 1205 and 144 human viral miRNAs. Hybridization signals were detected with the Agilent Microarray Scanner (Agilent, USA) and the scanned images were analysed using Agilent Feature Extraction Software (Agilent, USA). Data were acquired by first subtracting the background noise of raw data from hybridization images and then normalizing using the LOWESS filter (locally weighted regression).34 Spot (standard deviation)/(signal intensity) < 0.5. Differentially expressed miRNAs were identified by a cut‐off of fold change >1.5 and P < 0.01 by Student’s t test.

Calcified aortic valves and non-calcified valves from 3 CAVD patients were sent to carry on the miRNA microarray assay. Total RNA was extracted from tissues using the miRNAeasy Mini Kit (Qiagen GmbH). The miRNA microarray assay was performed by a service provider (LC Sciences). Total RNA (100 ng) was labeled with miRNA Complete Labeling and Hyb Kit (Agilent, USA) and hybridized on the Human miRNA Microarray Kit (Release 16.0, Agilent), which contains 60000 probes for 1205 and 144 human viral miRNAs. Hybridization signals were detected with the Agilent Microarray Scanner (Agilent, USA) and the scanned images were analyzed using Agilent Feature Extraction Software (Agilent, USA). Data were acquired by first subtracting the background noise of raw data from hybridization images and then normalizing using the LOWESS filter (locally weighted regression)^{45 (link)}. Spot (standard deviation)/(signal intensity) < 0.5. Differentially expressed miRNAs were identified by a cutoff of fold change ≥ 1.5 and P < 0.01 by student’s t-test.

MiRNAs extracted from the six MA, six PLF, and two CM samples were analysed using Agilent Human miRNA Microarrays (Agilent Technologies), which contain 1,226 human miRNAs and 146 human viral miRNAs. For miRNA detection, 100 ng RNA was labelled and hybridised using the Human miRNA Microarray kit (Rel 16.0; Agilent Technologies) according to the manufacturer's protocol (miRNA Complete Labeling and Hyb kit for miRNA Microarray System). Hybridisation signals were detected with a DNA Microarray Scanner G2505C (Agilent Technologies) and the scanned images were analysed using the Agilent Feature Extraction Software (v. 10.7.3.1). Data analysis was performed using Agilent GeneSpring GX software v. 11.0.2. (log₂ transformation). Baseline transformation was not performed.
The quality of miRNA extraction was analysed according to the variability in the number of microarray-detected miRNA species, signal intensity, and the correlation of miRNA expression among samples according to mean ± SD, coefficient of variation (CV), and correlation coefficient (CC).

miRNAs were labeled with miRNA Complete Labeling and Hybridization Kit (Agilent, USA) and hybridized at Human miRNA Microarray Kit (Release 16.0, Agilent, USA), which contained 60,000 probes for 144 human viral and 1205 human miRNAs. Microarray Scanner (Agilent, USA) was used to detect hybridization signals. Feature Extraction Software (Agilent, USA) was used for image analysis. Principal component analysis (PCA) and percentile normalization were performed using Gene spring 12.0. Cluster 3.0 was used for Cluster analysis and differentially expressed miRNAs were screened out.

Placental microRNA profiles were generated using Human miRNA Microarray kit, 8 × 60 K (based on miRBase release 16.0, Agilent Technologies). The miRNA Complete Labeling and Hyb Kit (Agilent Technologies) was used to label and hybridize 100 ng of total RNA, according to the manufacturer’s instructions. Briefly, RNA samples were dephosphorylated and labeled with cyanine 3-pCp by T4 RNA ligase for 2 h at 16 °C. RNA samples were hybridized to the miRNA microarrays for 20 h at 55° in a hybridization oven at 20 rpm. After washing, the slides were scanned by an Agilent Microarray Scanner (Agilent Technologies). The raw intensity of the array was scanned and extracted by BeadScan, and the data were corrected by background subtraction in the Genome Studio module.

Muscle tissues were used for miRNA isolation with a RNeasy Mini Kit (QIAGEN, Shanghai, China, #217004) according to the manufacturer's protocol. After determining the miRNA quality, 200 ng miRNA was used for microarray analysis with a Human MiRNA Microarray Kit (Agilent, Santa Clara, CA, #G4471A-029297) according to the manufacturer's protocol. The slides were scanned with an Agilent G4900DA SureScan Microarray Scanner System. The expression of 6 differentially expressed miRNAs, including miR-17-3p, miR-34a-3p, miR-107, miR-126-5p, miR-192-3p, and miR-532-3p, were examined by qRT-PCR using TaqMan miRNA assays. The assay IDs of these miRNAs were 477932, 478047, 478254, 480908, 478741, and 478336, respectively. The expression of these miRNAs in the same healthy control sample was defined as one-fold, and their expression in other samples was normalized to this control.