Sensifast probe lo rox

Total RNA was isolated from MCF7 cells using Trireagent (Ambion, Thermo Fisher Scientific, Carlsbad CA) and one microgram of RNA was reverse-transcribed using the High-capacity cDNA Reverse-Transcription Kit (Thermo Fisher Scientific, Carlsbad CA). cDNA was used for quantitative RT- PCR (qRT-PCR) analysis using ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) and SensiFAST Probe Lo-ROX (Bioline). Each amplification was performed in triplicate and the average cycle threshold (Ct) was used for analyses. Taqman assays (Thermo Fisher Scientific), chosen with the criterion of best coverage, were used. Genes analyzed and Assay IDs are listed in Supplementary Table 2.

Extracted RNA was quantified with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) with an assessment of A260/A280 (min > 1.7). 100 ng of RNA was used for reverse transcription assay (TaqMan^® MicroRNA Reverse Transcription Kit, Thermofisher) with primers for snU6, RNU43, miR-96-5p, miR-134-5p, miR-181b-5p and miR-200b-3p (TaqMan^®, Thermofisher). cDNA from previous reactions was used for quantitative Real-Time PCR (qPCR). qPCR was set up as it follows: 2 µl cDNA, 7,5 µl qPCR master mix SensiFAST Probe Lo-Rox (BIO-84020, Bioline, UK), 0,5 µl microRNA-specific TaqMan assays snU6, RNU43, hsa-miR-96-5p, hsa-miR-134-5p, hsa-miR-181b-5p, hsa-miR-200b-3p (Assay no.: 001973, 001,095, 000,186, 001,186, 001,098, 002,251) (TaqMan^®, Thermofisher, USA); molecular-grade water to final volume: 15 µl. U6 and RNU43 expression was used for miRNAs normalization between samples. Reactions were performed in triplicates. qPCR reaction was performed on Applied Biosystems^® 7500 Real-Time PCR System (Thermofisher, USA) with the following setup: 1 × 95 °C 5 min, 45 × cycles 95 °C 10 s and 60 °C 50 s.

cDNA was obtained as described earlier [20 (link)]. RNA expression was analyzed on cDNAs using the ViiA™ 7 Real-Time PCR System, SensiFAST™ Probe Lo-ROX (Bioline, Memphis, TN, USA), TaqMan gene expression assay according to the manufacturer’s instructions (Life Technologies, Waltham, MA, USA). mRNA quantification was expressed in arbitrary units, as the ratio of the sample quantity to the calibrator or to the mean values of control samples. All values were normalized to three endogenous controls: HPRT, GAPDH and β-ACTIN.
Primers for gene expression are listed in Supplementary Table S1. Gene expression of GLI1, HES1, c-MET, ABCG2, CD133, KRAS, BRAF, HPRT, GAPDH and β-ACTIN was assessed using Life technologies “best coverage” assays (Life Technologies, Waltham, MA, USA).

Total RNA was isolated from cells and human tissue using Trireagent (Ambion) and reverse transcribed in cDNA as previously described (12 (link)). cDNA was used for quantitative RT-PCR (qRT-PCR) analysis using ViiA ™ 7 Real-Time PCR System and SensiFAST™ Probe Lo-ROX (Bioline).
For each mRNA analysis 10 ng of cDNA were used. We selected best coverage TaqMan gene expression assay from Applied Biosystems and used according to the manufacturer's instructions. To analyze Numb isoforms, the following assay IDs were used: murine Numb p66 Mm01302754_m1; murine Numb p72 Mm01304901_m1, human Numb p66 Hs01105435_m1; human Numb p72 Hs01105426_m1.
mRNA quantification was expressed in arbitrary units and each amplification reaction was performed in triplicate. All Results were evaluated using the 2-ΔΔCT method and values were normalized to three endogenous controls: ß-actin, ß2-microglobulin, and Gapdh.

RNA was isolated from cells using TriReagent (Ambion) and retro transcribed using the High Capacity cDNA Reverse Transcription kit (Thermo Scientific). cDNAs were analyzed using the ViiA™ 7 Real-Time PCR System (Thermo Scientific) SensiFAST™ Probe Lo-ROX (Bioline), and TaqMan gene expression assays for βIIITubulin (Hs-00801390_s1, Mm00727586_s1) and GFAP (Hs-00909233_m1, Mm01253033_m1) for human and mouse respectively. Results were evaluated using the 2-ΔΔCT method and mRNA quantification was normalized to 2 endogenous controls: ACTIN and GAPDH.