Figure 5. Knockdown of p66Shc ameliorates H₂O₂-induced increase in p66Shc mRNA levels in early bovine embryos.

Following a combination of siRNA molecule microinjection and hydrogen peroxide (H₂O₂; 50 µM) treatments, total RNA was extracted from pooled groups of 5–8 cell embryos for p66Shc mRNA quantification. (A) Non-H₂O₂ treated embryos injected with negative siRNA exhibited significantly higher levels of p66Shc mRNA than non-injected controls. Embryos injected with the RNAi-E siRNA molecule exhibited significantly less p66Shc mRNA. H₂O₂ treated embryos injected with RNAi-E exhibited significantly lower levels of p66Shc mRNA. However, RNAi-E injected embryos treated with H₂O₂ exhibited significantly greater amounts of p66Shc mRNA than RNAi-E injected embryos that were not exposed to H₂O₂. (B) Histone H2A mRNA was quantified in parallel as internal controls for PCR efficiency. No significant differences in H2A mRNA were noted between treatment groups. Different letters above the histogram bars represent significant differences (P<0.05) in p66Shc mRNA abundance between groups.