I am currently working on a project where we are studying the effect of a drug on mineralization. I need to study the gene expression In vitro for RUNX, COL, BSP and Osterix between negative control (proliferation media), positive control (mineralization media) and treated group. I am using GAPDH as the housekeeping gene. I cultured 3T3-E1 pre-osteoblasts for 7,14,21, and 28 days and did RT-PCR at each time point. However, I am not able to see enough difference in gene expression folds between negative group and the other groups. I am seeing difference for BSP but not the other genes. Also, when I check for mineralization with Alizarin red, it works and I can see the difference in mineralization. Any ideas to why this is happening with PCR and how to fix it?