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. 2010 Jun 10;6(6):e1000979. doi: 10.1371/journal.pgen.1000979

Figure 5. REST–less breast cancer tumors display high levels of REST4 splicing and poor prognosis.

Figure 5

(A) Top panel: PCR primer screens for REST splicing in selected RNA from tumor samples indicated in Figure 2B: GSM124998, GSM125004, GSM125011, GSM125015, GSM125019, GSM125027, GSM125050, GSM125080 and GSM125088. Bottom panel: RNA was reverse-transcribed and PCR amplified with primers flanking the REST alternative intron/exon junction (REST primer set), showing alternative REST splicing only in the REST–less tumors. Additionally, selective PCR amplification of REST4 from tumor samples (using primers that target the REST4 50 bp exon (REST4 primer set)) demonstrated the presence of REST4 in the REST–less tumors, but not in any of the REST competent tumors. (B) Quantitative real-time RTPCR analysis of REST4 levels (relative to actin), in nine tumors represented in the microarray dataset GSE5460. REST4 mRNA, was detected in REST–less, but not RESTfl tumors after 35 cycles of amplification. (C) Patients with REST–less breast tumors in the superseries GSE6532, as defined by the 24-gene signature, show a significant decrease in their disease free survival with respect to their RESTfl counterparts (p<0.01).