qRT-PCR was performed for LRP2 and 18S (used as an endogenous control) with custom made primers (Life Technologies, Carlsbad, CA, USA) and iTaq Universal SYBR Green (Bio-Rad) on the Applied Biosystems ViiA 7 RT-PCR system.
Each sample's expression of LRP2 was first subtracted from its 18S expression to determine its [DELTA]CT.
siRNA Targeted to LRP2 in S1 Renal Tubular Epithelial Cells Reduces Relative mRNA Expression.
Four siRNA sequences targeting LRP2 were examined individually and as a pool for their efficacy in reducing LRP2 mRNA expression in mS1PT cells (Figure 1(a)).
In vivo siLRP2 outperformed siLRP2 sequence 4 in cell culture, eliciting a reduction of LRP2 mRNA expression to 15.6 [+ or -] 0.9% of siSCR treated expression (p = 7.2 x [10.sup.-8]).
In Vivo siLRP2 Reduces LRP2 mRNA Expression for Days in Renal Tubular Epithelial Cells.
While prolonged LRP2 mRNA suppression was achieved in renal cell culture, the degree of exposure time and concentration of the siRNA in cell culture constructs are unlikely to be achieved in vivo.
At 3 h, LRP2 expression was not significantly different between mice receiving PBS vehicle and those receiving 7.5 mcg/gm siLRP2 (Figure 3(b)).
At 6 h, LRP2 expression was reduced to 70.1 [+ or -] 6.3% compared to control (p = 0.015).
LRP2 Renal Expression Knockdown Is Not Dependent upon Administration Site.
Neither technique individually reached statistical significance as compared to control mice due to variability in control LRP2 expression.
In Vivo siLRP2 Is Effective at Reducing LRP2 mRNA Expression in Mouse Liver.