Sensifast sybr and fluorescein kit

Total RNA was isolated from cultured cells using RNeasy mini kits (Qiagen (#74104), Manchester, UK). An amount of 1 µg of total RNA was reverse transcribed to cDNA in a 20 µL reaction volume using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems (#4368814)). RT-PCR was carried out using the SensiFAST SYBR^® and Fluorescein Kit (Bioline (#BIO-96005)) in a final volume of 10 µL containing 0.5 µL cDNA and 250 nM primers and a Bio-Rad CFX 96 Real-Time Detection System. Primer sequences were 5′-TAACCTGGTCAGAAGTGTGCC-3′ (sense) and 5′-GGAGGGTTAACTTTTATACTCGGTGT-3′ (antisense) for IL-13Rα2 and 5′-CAGCCATGTACGTTGCTATCCAGG-3′ (sense) and 5′-AGGTCCAGACGCAGGATGGCATG-3′ (antisense) for beta-actin. The relative expression of IL-13Rα2 mRNA was calculated using the 2^−ΔΔCt method with beta-actin mRNA as the internal control [36 (link)]. To determine relative IL-13Rα2 mRNA expression in breast cancer tumors representing various stages of the disease, a similar RT-PCR methodology was applied to Origene TissueScan™ Breast Cancer cDNA Arrays (samples from CSRT104, BCRT103, and BCRT104; 60 tumor samples (n = 60) covering various disease stages with non-malignant (n = 7) breast tissue cDNA samples for comparison) [37 (link)].

Total RNA from cultured cells was isolated with Tri-reagent (Thermo Scientific) and using a phenol chloroform method. Contaminating DNA was digested using DNase I RNase-free (Thermo Scientific) treatment. Reverse transcription was carried out on 100 ng total RNA using Moloney Murine leukemia virus reverse transcriptase (M-MLV RT), provided with the Tetro Reverse Transcriptase kit (Bioline, London, UK). cDNA synthesis were performed following the manufacturers protocol, using a T100 Thermo Cycler (Bio-Rad, Hercules, CA, USA). Quantitative RT-PCR was performed in a total volume of 20 µL using the SensiFast SYBR and Fluorescein Kit (Bioline, London, UK). The PCR reaction profile consisted of initial enzyme activation at 95 °C for 10 s, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing 30 s. with temperature dependent on the primer sequences and extended at 72 °C for 30 s. with a single fluorescence measurement. The series of cycles were followed by a melt curve analysis to ensure reaction specificity. The expression level of each gene was normalized to the housekeeping gene, GAPDH. Subsequently, the relative gene expression (Qn) was calculated in relation to the GAPDH gene.

Total RNA was extracted from 24-h liquid cultures by grinding frozen tissue with a mortar and pestle followed by RNA purification using the RNeasy minikit (Qiagen). First-strand cDNA synthesis was performed using the Tetro cDNA synthesis kit (Bioline) in the presence of oligo(dT)₁₅ primer according to the manufacturer’s instructions. PCR was performed using a Bio-Rad iCycler and the Sensi-fast SYBR and fluorescein kit (Bioline). All amplification reactions included an enzyme activation step at 95°C (10 min), 40 cycles of denaturation at 95°C (15 s), annealing at 55°C (15 s), and an extension at 72°C (15 s) followed by a melt curve to ensure consistent product identity. Reactions were performed in triplicate for each sample with cDNA-specific primers for PkaC1 (PkaC1-RT-F and PkaC1-RT-R) and β-tubulin (5′ β-tubulin and 3′ β-tubulin; see Table S2). Additional reverse-transcriptase-negative control reaction mixtures were included for each sample and target to ensure amplification of cDNA only. Relative PkaC1 cDNA levels were quantified by subtraction of control β-tubulin threshold cycle (C_T) values from corresponding PkaC1 C_T values, followed by subtraction of these values from the average of the WT samples. 2 was then raised exponentially to this calculated value to determine the proportion of WT cDNA represented by each sample.