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)].
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