Mirvana rna isolation labeling kit

Following the overexpression of ESRRG in ASG or MKN45 cells for 3 days, the cells were harvested for RNA isolation using a mirVana™ RNA Isolation labeling kit (Ambion, Inc, Waltham, MA). The extracted total RNA (500 ng) was then used for labeling and hybridization to Human BeadChip V4 microarrays (Illumina, San Diego, CA) in accordance with the manufacturer’s protocols. After the bead chips were scanned with an Illumina BeadArray Reader, the microarray data were normalized using the quantile normalization method in the Linear Models for Microarray Data package in the R language environment. The expression level of each gene was then log₂ transformed before further analysis. The microarray data are available from the NCBI Gene Expression Omnibus public database (GSE78050).

Total RNA from human ovarian tumours was extracted using mirVana RNA isolation labeling kit (Ambion) according to the manufacturer's protocol. RNA purity was assessed by a Nanodrop spectrophotometric measurement (Thermo Scientific) of the OD260/280 ratio with acceptable values falling between 1.9 and 2.1. RNA was diluted to 50 μg μl⁻¹ using RNase-free water and 3 μl was used for miRNA profiling on the multiplexed nCounter Human v2 miRNA platform (NanoString Technologies, Seattle, WA, USA)53 (link). RNA samples were prepared by ligating a specific DNA tag (miR-tag) onto the 3′ end of each mature miRNA as per the manufacturer's instructions. Excess tags were removed by restriction digestion at 37 °C. Hybridizations were carried out by combining 5 μl of each miRNA-Tag sample with 20 μl of nCounter Reporter probes in hybridization buffer and 5 μl of nCounter Capture probes at 65 °C for 16–20 h. Excess probes were removed using a two-step magnetic bead-based purification on the nCounter Prep Station. Abundance of specific target molecules was quantified using the nCounter Digital Analyzer by counting the individual fluorescent barcodes and assessing target molecules. The data were collected using the nCounter Digital Analyzer and were analysed using the nCounter Analysis system as per the manufacturer's instruction.

Tumour spheroids created via co-culture with A549 and NIH3T3 cells were grown in a microfluidic system and harvested for RNA isolation at the indicated time point (Fig. 5a). Total RNA was extracted using a mirVana™ RNA Isolation Labeling Kit (Ambion, Inc, Waltham, MA). Total RNA (500 ng) was used for labeling and hybridization (Human Bead Chip V4 Microarray, Illumina, San Diego, CA), according to the manufacturer’s protocols. After scanning the bead chips using an Illumina BeadArray Reader, the microarray data were normalized using the quantile normalization method in the Linear Models for Microarray Data package in the R language environment. The expression level of each gene was log₂ transformed before further analysis.