Mircurytm power labeling kit

miRNAs expression profiling was performed using single-channel miRCURY LNA^TM miRNAs Array v16.0 (Exiqon, Inc., Woburn, MA, USA). miRCURY LNA^TM miRNAs Array v16.0 contains 1,891 capture probes, covering all human miRNAs annotated in the miRBase (release 16, http://mirbase.org/) and 66 new miRPlus^TM human miRNAs. The isolated total RNA of each subject was labeled using miRCURY^TM Power labeling kit (Exiqon, Inc., Woburn, MA, USA), and hybridized to miRCURY LNA^TM miRNAs Array v16.0 following Exiqon recommended standard protocol. Hybridization image scanning was performed by Axon GenePix 4000B microarray scanner (Molecular Devices, LLC., Sunnyvale, CA, USA). Scanned images were imported into GenePix Pro 6.0 software (Molecular Devices, LLC., Sunnyvale, CA, USA) for data extraction, median normalization and quality control. Our microarray data was MIAME compliant and has been deposited in the MIAME compliant database ArrayExpress (Accession number: E-MTAB-3514).

RNA samples were obtained from T2DM patients with CLI (n = 4) or without CLI (n = 4), matched for age, gender, and diabetes duration. The isolated total RNA from each subject was labeled using the miRCURY^TM Power labeling kit (Exiqon, Inc., Vedbaek, Denmark) and hybridized to the miRCURY^TM Array v16.0, according to the standard protocol. Hybridized arrays were analyzed by means of the GenePix 4000B microarray scanner (Molecular Devices, LLC., Sunnyvale, CA, USA), and the images were digitized with the Array-Pro image analysis software (Media Cybernetics, Silver Spring, MD, USA). Raw fluorescence data were processed using GenePix Pro 6.0 software (Molecular Devices, LLC., Sunnyvale, CA, USA). Individual spot fluorescence data were normalized based on the background and positive control fluorescence intensities.