Figure 3

The Isolated Bone RNA Supports Gene Expression Analysis using Real-time qRT-PCR. One μg of the isolated RNA was reverse transcribed using Multiscribe Reverse Transcriptase (Applied Biosystems) with random primers at 37°C for 2 hours. Gene expression of the bone specific transcription factor Runt-related transcription factor 2 (Cbfa1/Runx2) and the adipocyte-specific transcription factor Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) was analyzed via real-time PCR performed with TaqMan chemistry using the 7900 Real-time PCR system and universal cycling conditions. The Runx2 and PPARγ TaqMan primer-probe pairs were obtained from Applied Biosystems. (A) The amplification plot and (B) standard curve generated for Runx2. (C) Comparison of the gene expression of PPARγ and Runx2 from bone RNA isolated from the one-step approach versus multi-step methods. The bone was obtained from an animal fed a high fat diet for 4 months.