miRNA Complete Labeling and Hyb Kit

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

miRNA expression array
The analysis of the miRNA microarray was performed using a commercial human miRNA microarray (G4872A Human Rel. 16.0, Agilent Technologies, Santa Clara, CA, USA), which included probes for 1,205 human and 144 viral miRNAs based on Sanger miRBase Release 16.0. Total RNA samples from three independent, undifferentiated and decidualizing HESC cultures, were subjected to miRNA microarray. Using 100 ng of total RNA of each of the 6 samples, miRNAs were labelled and hybridised onto the miRNA microarray using the miRNA Complete Labelling and Hybridisation Kit (Agilent Technologies), according to the manufacturer’s protocol. After hybridisation for 22 h, the miRNA array was washed and the signal intensity of an individual miRNA probe spot was measured using a microarray scanner (G2505B; Agilent Technologies) and quantified with Feature Extraction ver. 10.7.3.1 software (Agilent Technologies) (Supplementary excel file 1). The 6 data sets were normalised using a quantile normalisation method33 (Supplementary Table S1). Expression of miRNAs was analysed pair-wise in undifferentiated and decidualising cells in the 3 biological repeat experiments. Upregulated and downregulated miRNAs were defined, respectively, by >2-fold or <0.5-fold change in expression level upon decidualization in pair-wise analysis of each of the 3 repeat experiments. The fold-change values of miRNAs were then calculated as average fold-change value from three independent paired cultures. P value was calculated by two-tailed Student’s t-tests.

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Manufacturer protocol

Download the product protocol from Agilent Technologies for miRNA Complete Labeling and Hyb Kit below.

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