RT-PCR analysis and expression profiles of the mRNA of H-FT and L-FT and RT-PCR analysis of L7 rRNA, all extracted from cells treated with AS-ODN against hours and L-FT.
SYBR-GOLD stains are of cycles 16 to 21 after a touchdown PCR step of 10 cycles, as described in “Materials and methods.” Below the stains are depicted the relative changes in L7-normalized H-FT and L-FT mRNA levels, together with their respective protein levels (measured by ELISA). Those changes were calculated using densitometric analysis of bands of cycle 18, which were within the linear range of increase in luminosity against cycle number. Means and standard deviations of 3 independent experiments are shown here. Cell incubations: untreated, 48 hours, no AS-ODN added; AS-ODN, 48 hours with 1 nM AS-ODN against H-FT (ASH-ODN), L-FT (ASL-ODN), or both (ASH-ODN+ASL-ODN); IN-ODN, 48 hours with 1 nM one ASH-ODN sequence in reverse order. See “Results” for ODN sequences. ASH-ODN and ASH-ODN+ASL-ODN treatments produced H-FT mRNA band densities that were significantly lower than those of the untreated cells (P ≤ .013 and P ≤ 2.8 × 10−4, respectively). ASL-ODN and ASH-ODN+ASL-ODN also significantly reduced the L-FT mRNA band densities (P≤ 9.87 × 10−5 and P ≤ .001, respectively). Statistics for the protein expression levels are compiled in Table 1.
