Evaluation of liver-specific VWF expression by RT-PCR after hydrodynamic injection of VWF cDNA. Analysis was carried out on total RNA from liver with VWF-specific primers (forward, 5′-GGACATTTTCTCAGACCACCATA-3′; reverse, 5′-TGTGGAGGCTGAGTTGGG-3′) 48 hours after hydrodynamic injection with murine VWF cDNA. To demonstrate that VWF RNA and not plasmid DNA was amplified, control reactions were performed without the addition of (A) reverse transcriptase. The lower panel shows identical reactions with (B) reverse transcriptase included. Lane 1, RT-PCR of RNA from untreated WT mice; lane 2, RT-PCR of RNA from untreated VWF^–/– mice; lane 3, RT-PCR of RNA from VWF^–/– mice 48 hours after hydrodynamic injection of 250 μg murine VWF cDNA; lane 4, RT-PCR of RNA from VWF^–/– mice 48 hours after hydrodynamic injection of 100 μg murine VWF cDNA; lane 5, RT-PCR of RNA from VWF^–/– mice 48 hours after hydrodynamic injection of 50 μg murine VWF cDNA; lane 6, RT-PCR of RNA from VWF^–/– mice 48 hours after hydrodynamic injection of 10 μg recombinant human VWF protein (rhVWF; negative control); lane 7, RT-PCR of RNA from VWF^–/– mice 48 hours after hydrodynamic injection of 250 μg eGFP cDNA (negative control); lane 8, RT-PCR of RNA from VWF^–/– mice 48 hours after hydrodynamic injection of saline (negative control). Using the described primers, the RT-PCR product of VWF mRNA is a 2.2-kb band, marked by an arrow in the left of both panels. RT-PCR products were visualized on ethidium bromide agarose gels.