miRCURY LNA™ microRNA Power Labeling Kits

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		Label all samples at one time to avoid day-to-day variations. The labeled samples can be stored at -80°C upto 3 months and successive hybridizations

Downstream tips
Label all samples at one time to avoid day-to-day variations. The labeled samples can be stored at -80°C upto 3 months and successive hybridizations

Publication protocol

Rats were anesthetized 1 or 3 days after surgery, and a 10 mm long segment of spinal cord, including the injury epicenter, was harvested and fresh-frozen in liquid nitrogen. Total RNA was isolated using TRIzol (Invitrogen, CA) and miRNeasy mini kit (Qiagen, West Sussex, UK) according to manufacturer's instructions. This efficiently recovered all RNA species, including miRNAs. RNA quality and quantity was measured using a nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA integrity was determined by gel electrophoresis.

After RNA isolation from the samples, the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer's guideline for miRNA labeling. One microgram of each sample was 3'-end-labeled with Hy3™ fluorescent label, using T4 RNA ligase according to the following procedure: RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon, Vedbaek, Denmark). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 5 min at 95°C. Then, 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3™), 2.0 μL of dimethyl sulfoxide (DMSO), and 2.0 μL of labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C.

After stopping the labeling procedure, the Hy3™-labeled samples were hybridized on the miRCURY™ LNA Array (v.16.0) (Exiqon) according to the manufacturer's instruction. The total 25 μL mixture from Hy3™-labeled samples with 25 μL hybridization buffer was first denatured for 2 min at 95°C, incubated on ice for 2 min, and then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization System (Nimblegen Systems, Inc., Madison, WI), which provided an active mixing action and constant incubation temperature to improve hybridization uniformity and enhance signal. Following hybridization, the slides were retrieved, washed several times using Wash buffer kit (Exiqon), and finally dried by centrifugation for 5 min at 400 rpm. Then, the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).

Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs with intensities ≥50 in all samples were chosen for calculating a normalization factor. Expressed data were normalized using median normalization. After normalization, miRNAs that had significant differential expression were identified through Volcano Plot filtering. Hierarchical clustering was performed using MEV software (v4.6, TIGR).

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Manufacturer protocol

Download the product protocol from Exiqon for miRCURY LNA™ microRNA Power Labeling Kits below.

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