Northern blot analysis of SFV RNAs in transfected cells.

<p>BHK-21 cells were transfected with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1) in the presence of 300 picomol of each miRNA inhibitors (designated with “+”) or 900 pmol of irrelevant control oligonucleotide (designated with “−”). Total RNA was isolated from cells at 12 h p.t. or 36 h p.t.. RNA (5 µg) was loaded on a 1.4% denaturing agarose gel, resolved by electrophoresis and visualised by northern blotting with a DIG-labelled RNA probe complementary to the 3′ UTR of SFV4 or to β-actin mRNA. Arrows left of the panel designate viral genomic RNA (A), SG mRNA made from the native SG promoter (B), additional SG RNA synthesised from the duplicated SG promoter in SFV4-2SG-Gluc (C) and β-actin mRNA, used as loading control (β-act). The panel is composed of pictures obtained by two different exposures of the same filter, which was necessary due to the huge difference in viral RNAs levels at 12 and 36 h p.t.. After the shorter exposition (36 h p.t.), mRNA of β-actin is hardly detectable. Longer exposure (12 h p.t.) detects mRNA of β-actin in mock-samples at 36 h. p.t., but it is masked by the strong signal from viral RNAs in samples from cells transfected with DNA/RNA layered SFV replication-competent vectors. The experiment was repeated twice with similar results.