Ambion mirvana rna isolation kit

DiL-Ac-LDL-labelled HUVEC and CFSE-labelled MDA-MB-231 cells were co-cultured in Matrigel using standard co-culture protocol for 36 h. The cells were harvested and stained with PECAM-1 (1:25) (Abcam) and Alexa Fluor 647 (1:100) (Life Technologies) secondary antibodies. Stained cells were then FACS sorted into PECAM-1 + /DiL-Ac-LDL + /CFSE + and PECAM-1 + /DiL-Ac-LDL + /CFSE − populations, capturing HUVECs that did and did not receive nanobridge-mediated transfer. The sorted cells were then pelleted and snap frozen in liquid nitrogen. Total RNA isolation was carried out using the Ambion mirVana RNA Isolation Kit (Life Technologies) as per the manufacturer’s protocol and quantified using NanoDrop (Thermo Scientific). An miRNA microarray was carried out on two sets of the above mentioned samples from independent experiments with HUVECs stained with 7 μM CFSE as control. The Affymetrix GeneChip miRNA 3.0 Array was used. Labelling was carried out using the Affymetrix Flashtag Biotin HSR RNA labelling kit and standard protocol with a 1:500 dilution of ATP for Poly(A) Tailing. Hybridization was carried out using the Affymetrix GeneChip Hybridization Oven 640 for 42 h. Microarray data analysis was carried out using the bioinformatics toolbox in MATLAB (MathWorks); GEO accession number GSE72679.