Figure 2

The presence of steady-state SPAM1 mRNA as detected by LM/RT-PCR of the lysate recovered from human epididymal epithelial cells (H EPI, test) and stromal cells (H STR, control). RNA was subjected to first strand synthesis with (+) or without (-) reverse transcriptase (RT), followed by PCR amplification. The expected 320 bp band for the SPAM1 cDNA fragment is seen in A) for Subjects 1–3 in Lanes 2, 4, 6, for RT(+) reactions. This band was verified to be SPAM1 by sequencing. No 320 bp band is detected in Lanes 3, 5, 7, in the absence of RT showing that the SPAM1 bands are not from DNA contamination. In Lanes 8 and 9 no SPAM1 bands are detected for the stromal cells, but one is seen in Lane 10 for genomic DNA, the positive control. In Lane 11, no sperm-specific band for PRM1 cDNA could be amplified from the cDNA of epithelial cells of Subject #1, verifying the homogeneity of the cells. In Lane 12 the 110 bp band (arrowed on the right) is the PRM1 DNA fragment amplified from genomic DNA, as a positive control. A band of the same size should be generated from the cDNA in the presence of mRNA from the lysate. Note that the primer cloud for SPAM1 is not present for PRM1 in Lanes 11 and 12. In B) the 320 bp band in Lanes 2 and 4 are from the corpus of Animal #1 and the cauda of Animal #2, respectively. They were also verified to be SPAM1 by sequencing. The marker, 100 bp ladder, is shown in Lane 1 of both A) and B). The same results were obtained for replicate experiments.