Rnaqueous 96 isolation kit

Total RNA from MCF-7 and ZR75.1 cells was prepared using the RNAqueous-96 Isolation Kit (Ambion). Total RNA from primary breast tumors was prepared as described in [37 (link)]. Total RNA was subjected to reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR amplifications of IKKε, NOX2, NOX5, S9 and β-actin genes were performed using the Fast start SYBR Green Kit (Roche) in a Rotor-Gene 3000 Real-Time Thermal Cycler (Corbett Research). Absence of genomic DNA contamination was analyzed using a reaction without reverse transcriptase. Gene expression normalized over actin or S9 was analyzed using the ΔΔ Cycle threshold (C_t) method [38] (link) or as absolute mRNA copy numbers using plasmid-based standard curves as described in [39] (link). RT-PCR analysis of NOX/DUOX isoforms expression was performed as previously described [35] (link). Positive controls were used for each gene, as follows: total RNA from colon was used for NOX1; total RNA from DMSO-differentiated HL60 was used for NOX2; total RNA from human fetal kidney was used for NOX3; total RNA from MRC-5 were used for NOX4; total RNA from human spleen was used for NOX5; total RNA from human thyroid was used for DUOX1 and DUOX2. Human thyroid total RNA, human fetal kidney total RNA, human colon total RNA, and human spleen total RNA were purchased from BD Clontech.