7900ht thermal cycler apparatus

RNA purification and quantitative real time RT-PCR (qPCR) were used to measure hippocampal α and β estrogen receptor levels. In the DREADDs experiment, CNO injections were performed 70 min before sample collection, considering 40 min for DREADDs activation and further 30 min for RNA transcription. Total RNA was isolated from hippocampus brain punches using Total RNA purification Kit (Norgen Biotek, Thorold, Canada). RNA quantity was determined by absorbance at 260 nm using a NanoDrop UV–Vis spectrophotometer. For the reverse transcription of mRNAs (ER α/β and endogen control), random complementary DNA sequences were obtained using the High Capacity Reverse Transcription Kit (Applied Biosystems, Branchburg, NJ, USA). cDNA templates (8 ng for mRNA sample) were amplified by qPCR with the Taqman technology, using the 7900HT thermal cycler apparatus equipped with the SDS software version 2.3 (Applied Biosystems) for data collection. For ERα (Taqman assay: Mm00433149_m1) and for ERβ (Taqman assay:Mm00599821_m1) Ct values were normalized to averaged measures of Tata Binding Protein (TBP, Taqman assay ID Mm00446973_m1) [27 (link)]. All data were run in triplicate and were expressed as Fold Changes versus the control group, according to the ΔΔC(t) method [28 (link)].

Complementary DNA was obtained using the High Capacity Reverse Transcription Kit (Applied Biosystems, Branchburg, NJ, USA). Complementary DNA templates (10 ng) were processed with quantitative PCR using the 7900HT thermal cycler apparatus equipped with the SDS software version 2.3 (Applied Biosystems) for data collection. Taqman primer sets (Applied Biosystems, see Supplementary Table S1) were used to amplify mouse SIRT1–7, as well as human SIRT1 and C_t values were normalized to measures of TBP (Tata Binding Protein) and Pgk1 (Phosphoglycerate kinase1) mRNAs for mouse tissues, or Tata Binding Protein and GUSB (Glucuronidase beta) mRNA for human PBMC. All data were run in triplicate and analyzed using the ΔΔC(t) method.^{34 (link)}