C - Michigan Technological University

varied the length of bear serum stimulation and the differentiation state of the MC3T3-E1cells. After stimulation in one of four conditions, RNA was isolated, and real-time PCRperformed as described in section 5.2.5. In each condition, three potential referencegenes (β-actin, ubiquitin, cyclophilin) and the geometric means [305] of the variouscombinations of reference genes were checked for seasonal trends by a single-factorANOVA for season, blocking by bear. The reference gene (or geometric mean) with thelowest magnitude of seasonal variation and the highest least significant number waschosen for normalization. Ubiquitin was later removed from reference gene analysis inall samples since it had a significant seasonal trend in one of the conditions.In the first condition, undifferentiated pre-osteoblasts (seeded the previous day)were stimulated in 2% seasonal bear serum for 3 hours. Since bear serum was collectedevery 10 days, several prehibernation, hibernation, and post-hibernation serum sampleswere available for each bear. Each well received only one serum sample, and the bearand date of collection were recorded. After 3 hours of stimulation, RNA was isolated.Real-time PCR was performed on BAD, Bcl-2, Cyclin D1, Per1, PTH1R, Runx2, Smurf1,and ubiquitin. Cyclophilin was used as a reference gene. In the second condition, aftercells were allowed to attach overnight, they were switched to differentiation media (α-MEM with 10% FBS, 50 μg/mL ascorbic acid and 10 mmol β-glycerophosphate) andgrown for 6 days, with a media change on the third day. Differentiating osteoblasts werethen stimulated in differentiation media supplemented with 2% seasonal bear serum for3 hours. Real-time PCR was performed on Akt, BAD, BAK, BAX, Bcl-XL, c-fos, MCSF,OCN, OPG, p53, Per1, TLR4, and ubiquitin. Samples were normalized to the geometricmean of 2 reference genes (β-actin and cyclophilin). In the third condition, after cellswere allowed to attach overnight, undifferentiated pre-osteoblasts were stimulated innormal growth media supplemented with 2% bear serum for 24 hours. Real-time PCRwas performed on ADRβ2, Akt, BAD, BAK, BAX, Bcl-2, Bcl-XL, BmalI, ColI, Cyclin D1,M-CSF, OCN, OPG, OPN, p53, Per1, Per2, PTH1R, RANKL, Runx2, Smurf1, TLR4, andubiquitin. Samples were normalized to the reference gene β-actin. In the fourthcondition, after cells were allowed to attach overnight, media was replaced withdifferentiation media containing 2% bear serum (instead of FBS). Cells were allowed todifferentiate in bear serum for 6 days, with a media change on day 3. Real-time PCRwas performed on BAD, BAX, Bcl-2, c-fos, ColI, Cyclin D1, OCN, OPG, OPN, Per1,94