Inhibition of EGFP expression and detection of expressed shRNA from bovine and mouse U6 promoters. (1) transfection free negative control (cells only); (2) transfection of pEGFP-N1; (3) cotransfection of pEGFP-N1 and pBovineU6-shScrambled; (4) cotransfection of pEGFP-N1 and mouseU6-shEGFP; (5) cotransfection of pEGFP-N1 and pBovineU6-shEGFP. All transfections were performed in triplicate. (A): Typical fluorescence images of Vero and MDBK cells (Magnifications ×100). (B): Mean Fluorescence Intensity (MFI) of Vero and MDBK cells 72 hours post-transfection determined by flow cytometry. MFI of EGFP expressed as a percentage of the MFI of pEGFP-N1 + pBovineU6-shScrambled. (C): Detection of EGFP shRNAs in Vero cell extracts. Small RNAs were isolated from Vero cells and hybridised to a radiolabelled RNA probe identical in sequence to the EGFP shRNA (shEGFP). shEGFP was detected in lanes 4 and 5 only, and miR-16 was used as a loading control.