Semiquantitative Cardiac-Specific Gene Expression Validation of the DNA Methylation Microarray in Human Mesenchymal...

Epigenetic events such as DNA methylation and histone modifications affect a wide range of genomic functions without impacting the nucleotide sequence. Aberrant DNA methylation has been related to a variety of disorders, including cancer, obesity, and addiction. DNA methylation is defined by the presence of a methyl group at the fifth position of the cytosine pyrimidine ring, which occurs in roughly 80% of CpG dinucleotides in the human genome. It has also been linked to functional significance and is referred to as the genome's "fifth base." Studies speculate the use of epigenetic modifiers like 5'azacytidine (5-AC) in MSCs demethylates DNA, resulting in the induction of cardiac-specific genes. However, the restricted expression of the CSG set results in inadequate differentiation of WJ-MSCs into CMs. This chapter covers in detail the semiquantitative real-time validation approach that uses the relative expression quantification method with SYBR Green. We adopted the master regulatory cardiac specific gene (GATA4), which is critical in the phenomenon of cardiac lineage differentiation for the real-time validation. Further, this chapter describes the technique used to validate bisulfite specific PCR analysis, real-time corroboration of methylation microarrays, including total RNA extraction, quality control, cDNA synthesis, and real-time validation utilizing the relative quantification approach.