Taqman universal pcr master mix assays

Seven genes, described to be involved in epigenetic regulation, were selected for the analysis of their expression profile in the thalamus of Tg338 mice: Dnmt1, Dnmt3a, Dnmt3b, Hdac1, Hdac2, Tet1, and Tet2. Table 2 shows TaqMan assays (Thermo Fisher Scientific) used for the amplification of the genes of interest.
Total RNA was isolated using the RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA, USA). The quality and quantity of RNA were determined using a NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA, USA). An amount of 200 ng of total RNA was used for retrotranscription with qScript^TM cDNA Supermix (Quanta Biosciences^TM, Gaithersburg, MD, USA), according to the manufacturer’s instructions. The resulting complementary DNA (cDNA) was diluted 1:5 in water and gene expression was quantified by RT-qPCR using the TaqMan universal PCR master mix assays (Thermo Fisher Scientific) in a StepOne Plus Real-Time PCR instrument (Applied Biosystems, Waltham, MA, USA). All reactions were run in triplicate. The comparative quantification of the results was standardized by the 2^−∆∆Ct method [78 (link)], using the geometric mean of Sdha and H6pd housekeeping genes (Table 2) as a normalizer. Student’s t-test was applied to identify differences between groups, which were considered significant at p < 0.05.