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. 2014 Jul 3;9(7):e101629. doi: 10.1371/journal.pone.0101629

Figure 4. EGFP RNA is present in spermatozoa of mice inoculated with EGFP-infected A-375-cells.

Figure 4

A: Murine protamine 2 gene (Prm2) amplification products used to select DNA-free RNA samples. Exemplifying gel of Prm2-specific PCR amplification products of intron-containing DNA from the mouse sperm genome (lane 1) and RT-PCR products from RNA extracted from spermatozoa of non-inoculated (lane 2) and EGFP-expressing A-375+ inoculated (lane 3) mice, both showing the spliced Prm2 form. B: Southern blot hybridization of RT-PCR amplified RNA from: spermatozoa of non-inoculated control (lane 2) and A-375+ inoculated (lane 3) mice, and from non-infected (lane 4) and EGFP-infected (lane 5) A-375 whole cells. Hybridization was carried out with an EGFP-specific internal probe. Lane 1 is a no-RNA control. The bottom panel shows RT-PCR amplification products from the same samples using GAPDH-specific primers as a loading control. C: Southern blot hybridization of RT-PCR amplified RNA from spermatozoa from a control mouse (lane 2) and from a single EGFP-expressing A375+ inoculated mouse (lane 3); lane 1 shows a no RNA reaction. As in B, the bottom panel shows GAPDH amplification from the same samples. D: RT-PCR amplification with (+RT) or without (-RT) reverse transcription step of RNA extracted from sperm-depleted epididymis from two inoculated EGFP mice. No EGFP-specific amplification products were visible by ethidium bromide staining (EtBr) nor by Southern blot hybridization (Hyb) using an EGFP radioactive probe. The bottom panel shows GAPDH amplification from the same samples.