Research Paper Volume 11, Issue 10 pp 2968—2997

Figure 8. Examination of endo-siRNA target gene knockdown. To confirm the functional significance of the endo-siRNA, RNA9878, GV oocytes were microinjected with a synthetic RNA9878 small RNA mimic or a non-targeting negative control. (A) To confirm successful microinjection of the RNA9878 mimic, the expression of RNA9878 was assessed via RT-qPCR immediately after injection. At 24 h post-injection, the relative levels of (B) Kifc1 and (C) Kifc5b were assessed in non-targeting and RNA9878 mimic injected oocytes via RT-qPCR. The U6 small nuclear RNA and Ppia were employed as endogenous controls to normalize the expression levels of the target endo-siRNA and mRNAs, respectively. (D) Non-targeting and RNA9878 mimic injected oocytes were then fixed and the expression of HSET was examined. Oocytes were labelled with anti-HSET antibodies followed by goat anti-rabbit 633 Alexa Fluor-conjugated (grey) secondary antibodies. Oocytes were then counterstained with the nuclear stain Hoechst 33342 (blue) and viewed using confocal microscopy. Scale bar = 20 μm. RT-qPCR experiments were performed in technical and biological triplicate, with each biological replicate comprising 10 oocytes randomly sampled from a pool of oocytes isolated from three animals. Similarly, immunofluorescence experiments were repeated on three biological replicates, with each replicate comprising a minimum of 10 oocytes randomly sampled from a pool of oocytes isolated from three animals. Values are shown as a mean of each replicate ± SEM. Statistical analyses were performed using Student’s t-test, * p < 0.05, ** p < 0.01. AU, arbitrary units.