RNA Extraction and Real-time qPCR Analysis

Total RNA from MDA231 and MDA231-BrM2 cells was extracted using the miRNeasy Micro Kit (cat# 217084, Qiagen, Germany). A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) was used to evaluate RNA purity and concentration using two absorbance ratios (A260/A280 and A260/A230). For single-stranded cDNA synthesis, the RT² First Strand Kit (cat#, 330401, Qiagen) or the miScript II RT Kit (cat#, 218161, Qiagen) was used as per the manufacturer’s protocols. Real-time quantitative PCR was performed on the “Applied Biosystems^® StepOne™ Real-Time PCR System” with the RT² SYBR Green ROX qPCR Mastermix (cat# 330522, Qiagen) for mRNA expression and the miScript SYBR Green PCR Kit (Cat# 218075, Qiagen) for miRNA expression using the “Applied Biosystems^® StepOne™ Real-Time PCR System”.13 (link) hsa-miR-623 and MMP1 relative expressions were normalized to the housekeeping gene U6 small nuclear RNA and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), respectively, and the data were analyzed using the 2^–ΔCt and 2^–ΔΔCt methods.34 (link) The primers used were obtained from Qiagen: Hs_miR-623_1 miScript Primer Assay (Qiagen), Hs_RNU6-2_11 miScript Primer Assay (cat# MS00033740), RT² qPCR Primer Assay for Human MMP1 (cat# PPH00120B-200), and RT² qPCR Primer Assay for Human GAPDH (Cat# PPH72843A).