| Popis: |
The retinoblastoma protein (pRb) plays a central role in the regulation of cell cycle by interaction with members of the E2F transcription factor family. As a tumor suppressor protein, pRb is frequently dysregulated in several major cancers. In addition to mutations, inactivation of pRb is also caused by epigenetic mechanisms including alterations of DNA methylation. There are three CpG islands located within the RB1 gene that encodes pRb that are closely associated with the regulation of pRb expression. Aberrant DNA methylation at the RB1 gene has been reported in sporadic retinoblastoma as well as other cancers including glioblastoma, hepatocellular carcinoma, and breast cancer. Recent studies have revealed that the RB1 gene is imprinted. Therefore, quantitative analysis is required to detect aberrations in DNA methylation associated with imprint deregulation. Pyrosequencing® is considered as the method of choice for quantitative and reproducible analysis of DNA methylation with single base resolution. In this chapter, we provide a detailed protocol for the quantitative analysis of RB1 gene methylation using bisulfite Pyrosequencing®. |
