Variable IGF1R mRNA Expression in Individual GV and In Vitro Matured M2 Human Oocytes
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Single GVs were placed directly into lysis buffer (5μl). Oocytes in vitro matured (IVM) to the M2 stage were placed into individual tubes containing lysis buffer after 24 to 48 hours (h) in culture. Total RNA isolated from cumulus granulosa cells (GC) using the TRIzol reagent (Invitrogen) as stated in the manufacturer’s protocol served as a positive control. Reverse Transcription (RT) was performed using anchored oligo-dT primers (IDT, Coralville, IA) and Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen) on the entire 5μl oocyte lysate at 37°C for 1.5h or 1mg total GC RNA 42°C for 1h. Quantitative polymerase chain reaction (qPCR) was performed combining standards or 1μl cDNA with PCR buffer containing SYBR Green I (Sigma Chemical Co, St. Louis, Missouri), Taq polymerase (Genscript, Piscataway, NJ), and primers for IGF1R. Melting curves were routinely determined to ascertain generation of only the expected product. |
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