Rna extraction protocol

Immature (21 d old) and mature (6–8-week-old) WT and gcAdarKO mice were superovulated as described above, at specific time points post-hCG administration mice were euthanized, and the ovaries were dissected free of the ovarian bursa and then rinsed in sterile dPBS to remove blood and tissue debris. Ovaries were then placed in dishes of FHM with 4 mg/ml BSA. Large antral follicles visualized under a Nikon SMZ1000 with a Nikon NI-150 high intensity illuminator were individually punctured with insulin syringes (28 gauge) to allow expulsion of mGCs and cumulus oocyte complexes. Smaller follicles were left with the remaining ovarian tissue which was removed from the dish before naked oocytes and cumulus oocyte complexes were removed and discarded. Mural GCs and the surrounding media was then collected, placed into a 1.5 ml Eppendorf tube and gently pelletized at 800× g, excess media was aspirated using a pipette, and 500 µL of TRI Reagent (Invitrogen, Waltham, MA) was applied to the cell pellet. Solubilized cell pellets were frozen at −80 °C overnight followed by completion of the manufacturer’s RNA extraction protocol (Invitrogen).

Cultured AML and MDM cells were harvested and RNA was isolated using the manufacturer’s RNA extraction protocol (Invitrogen). cDNA was prepared. Real-time PCR was performed using predesigned TaqMan human primers in the Applied Biosystems 7500 Real-Time PCR System. Expression levels of basal mRNA in AML and MDM cells were normalized to actin beta (ACTB) and calculated by the ΔΔ threshold cycle (ΔΔCT) method. The detailed protocol is given in the detailed supplemental methods section.