Methylation-Specific PCR In Situ Hybridization

Methylation-specific polymerase chain reaction (PCR) in situ hybridization, using paraffin-embedded, formalin-fixed tissues or formalin-fixed cell preparations, allows one to determine which specific cells have silencing of a given gene owing to hypermethylation of the promoter. Standard in situ hybridization, after conversion of nonmethylated bases, would not have sufficient sensitivity to detect the one or two copies of the promoter region of interest. The framework of the methodology is the same as solution-phase methylation-specific PCR. However, when working with intact tissue or cell preparations, adequate formalin fixation and protease digestion are essential for satisfactory results. Furthermore, since one often uses an oligoprobe, probe size (at least 40 bp), labeling method (3′ end tailing), probe concentration, and posthybridization stringency conditions can all have an impact on the final results.