To analyze TRH gene methylation at the cg01009664 position using the pyrosequencing assay, we first tested the assay in normal and cervical cancer cell lines, which showed low and high TRH methylation levels, respectively, as shown in Figure 1B. Pyrogram of five CpGs of CaSki, SiHa and C33A cervical cancer cell lines showed high methylation levels (80–100%) while normal cervical cell lines revealed low methylation levels (<10%) as shown in Figure 2. The gene discussed is TRH; the disease is cervical cancer.