Single tube taqman mirna assay

The detailed procedure for RNA isolation from cells collected by the LCM technique has been described elsewhere [26 (link)]. RNA was extracted from LCM–collected samples of the tumor and neighboring non-tumor breast cells of each patient using a mirVana miRNA isolation kit (Ambion, Austin, TX, USA). The RNA concentration was estimated with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). An appropriate probe and primer set was used to detect the expression of genes encoding hsa-miR-139 (AB assay ID: 17100) and then subjected to the single-tube TaqMan miRNA assay (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems instrument. The results were normalized against RNU6B. Fold-changes in expression were calculated by relative quantification using the 2^−ΔΔCT method with three independent experiment.

RNA extraction, reverse transcription (RT), and quantitative real-time amplification (q-RT-PCR) were performed as previously described [32 (link)]. Briefly, total RNA was isolated from MM cells by using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. The integrity and purity of total RNA isolated were measured by using Nanodrop (Celbio Nanodrop Spectrophotometer nd-2000, Thermo Fisher Scientific, Waltham, MA, USA).
The single-tube TaqMan miRNA assay (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) was used to detect and quantify mature miR-29b (Assay ID: 000413, Applied Biosystems, Life Technologies) according to the manufacturer’s instructions, by the use of QuantStudio 12K Flex reader (Thermo Fisher Scientific, Waltham, MA, USA). miR-29b expression was normalized on RNU44 (Applied Biosystems, assay ID: 001094).

Total RNA was extracted from cells using TRIzol^® reagent (Gibco, Life Technologies, Carlsbad, CA), following the manufacturer’s instructions. The RNA quantity and quality was assessed through NanoDrop^® (ND-1000 Spectrophotometer). To evaluate gene expression levels, 1000 ng of total RNA were reverse transcribed to cDNA using the “High Capacity cDNA Reverse Transcription Kit” (Applied Biosystems, Carlsbad, CA). The single-tube TaqMan assays (Applied Biosystems, Carlsbad, CA) were used to detect and quantify genes: EZH2 (Hs01016789_m1), MALAT1 (Hs00273907_s1) according to the manufacturer’s instructions, using Viia 7 Dx multicolor detection system (Applied Biosystems, Carlsbad, CA). The obtained Threshold Cycle (CT) values were normalized on GAPDH (Hs03929097_g1). The single-tube TaqMan miRNA assay (Applied Biosystems, Assay id 000413) was used to detect and quantify mature miR-29b expression, that was normalized on RNU44 (Applied Biosystems, Assay id 001094). Comparative real-time polymerase chain-reaction (RT-PCR) was carried out in triplicate, including no-template controls. Relative expression was calculated using the comparative cross threshold (Ct) method.