Detection of BORIS mRNA in breast cell lines, primary tumors, and controls.

Three endpoint RT-PCR assays, spanning different intron splice junctions, were designed to detect mature transcripts and alternatively spliced variants of BORIS mRNA (2–11, 2–6, 6–10; Figure 1). These RT-PCR reactions were performed using RNA derived from cell lines, breast tumors, normal breast tissue, or several different positive and negative controls (described in text). Cell lines with evidence of 20q13.2 amplification [23] are indicated by an asterisk (*). The detection of BORIS mRNA using the different end-point PCR assays is indicated by a “+”, whereas the lack of a product is indicated by a “-”. NP = not performed. A quantitative real-time RT-PCR (qRT-PCR) assay, spanning intron 10 (10–11), was used to quantify BORIS mRNA levels. The median threshold cycle (Ct; the PCR cycle for which products are detected above baseline), of triplicate reactions is reported for both BORIS (10–11) and TBP. BORIS mRNA was not detected (ND) in most cell lines and all tumors. Quantitative results were normalized and are expressed as a percentage of TBP expression (% TBP). For the N697 sample, only TBP end-point PCR was performed and it was positive (#). MDA-MB-435 is considered to be melanoma-derived (†).