Figure 2

5'RLM-RACE procedure in DU-145 and LNCaP prostate cancer cell lines. A) Two forward primers used for the two rounds of RACE-PCR recognizing the adapter sequence, and two reverse primers, against the fifth and third exon of SHBG, are shown. B) In the DU-145 5' RACE, one major band (asterisk) was obtained. TAP+ samples include total RNAs treated with Tobacco Acid Pyrophosphatase, and TAP-samples are negative controls that include RNAs that did not incorporate the RNA Adapter oligonucleotide. Ctrl-: Negative controls performed using water instead of cDNA. C) Two cloned products were obtained from the major band: RACEfrag 1 and RACEfrag 2. The empty vector (E.V.) provided an amplification product of 200 nucleotides, corresponding to the M13 flanking sequence. D) In the LNCaP 5' RACE, one major band (asterisk) was obtained. E) The cloning of the major band generated RACEfrag 3 and RACEfrag 4 products. Insert and M13 flanking sequence size are indicated in the four RACEfrags.