Quantifying TERT Promoter Methylation in Thyroid Cancer

Methylation density at the TERT promoter was quantified in MTCs, normal thyroid samples and MTC cell lines by Pyrosequencing. Genomic DNA (150 ng) was subjected to sodium bisulfate modification using the EpiTect Bisulfite Kit (Qiagen AB, Sweden) according to the recommendations of the manufacturer. Converted DNA was then used as template for PCR amplification at 58°C annealing temperature. A Pyrosequencing assay targeting eight CpGs in the TERT promoter (Figure 1) was designed using PyroMark Assay Design software version 2.0 (Qiagen). The assay included the following primers: 5′-GGGTTTGTGTTAAGGAGTTTAAGT-3′ (forward biotinylated); 5′-AAACCCAAAACTACCTCCA-3′ (reverse); and 5′-CCAAAACTACCTCCAAAT-3′ (sequencing). The PCR products were immobilized to streptavidin-coated Sepharose beads and were captured using a pump with filter. Pyrosequencing reactions were run in a PyroMark Q24 and the data were analysed with the PyroMark Q24 software (Qiagen AB, Sweden). For each sample, a methylation index (Met I) was calculated as the mean methylation level of all CpG dinucleotides covered [28 (link)]. The methylation status in MTCs was defined based on the comparison to the normal thyroid samples.