7900ht sequence detection system

Total RNA was extracted from liver and skin tissues using the RNeasy^® Mini Kit (74106, QIAGEN, Hilden, Germany). RNA was reverse-transcribed using the SuperScript VILO cDNA Synthesis Kit (11754, Thermo Fisher Scientific, Waltham, MA, USA). Diluted cDNA was used as a template for quantifying relative mRNA concentrations. RT-qPCR was performed using Taqman Gene Expression master Mix (Thermo Fisher Scientific, Waltham, MA, USA), Taqman probes (Table S1), and the 7900HT Sequence Detection System (Thermo Fisher Scientific, Waltham, MA, USA). The primers used are listed in Table S1. Relative gene expression values were normalized to B2M for the liver and Polr1a for the skin, using the comparative Ct (threshold cycle) method.

Genomic DNA was extracted from buccal cells following the procedure described by Freeman et al. (2003) (link). The BDNF Val66Met polymorphism was assayed using the rs6265 TaqMan SNP Genotyping Assay (Thermo Fisher Scientific, United States) and analyzed on a 7900HT Sequence Detection System (Thermo Fisher Scientific, United States).

Total RNA was extracted and reverse transcribed as reported in [29 (link)]. The mixture was amplified by a 7900HT Sequence Detection System (Thermo Fisher Scientific, Waltham, MA USA). Actin was used as the internal control. Primers and TaqMan probes were purchased for all genes as ready-to-use solutions (Assay on Demand, Thermo Fisher Scientific, Waltham, MA USA). mRNA relative expression levels were calculated with the 2-ΔΔCt method. Wt for wt_bez and G12C for G12C_bez clones were set to 1. Two samples that showed at least a 2-fold difference in expression were considered differently expressed.

Genotyping for each mouse was performed by PCR on the condition: PCR was performed in 12.5 µl of 2×Taq PCR MasterMix (kt201, Tiangen, Beijing, China), 1 µg DNA, 0.1 µM forward primer and reverse primer, and 23 µl ddH2O. PCR reactions were run on a 7900 HT Sequence Detection System (Thermo Fisher Scientific, Waltham, MA, USA). Cycling conditions were 94 °C for 3 min, followed by 30 cycles of 94 °C 30 s, 55 °C 30 s, 72 °C 60 s, and a final cycle at 72 °C for 5 min. Five µl of PCR products were loaded on 1% of agarose gels for electrophoresis. Mice did not fit the genotype criteria (Brca1+/−, or Brca1+/− Trp53+/−) were excluded.
The PCR primers used for genotyping Brca1 (Table S1):
Primer 1- F1: 5′-CTGGGTAGTTTGTAAGCATCC-3′,
Primer 2- R1: 5′-CAATAAACTGCTGGTCTCAGGC-3′,
Primer 3- R2: 5′-CTGCGAGCAGTCTTCAGAAAG-3′
The PCR primers used for genotyping Trp53:
Primer 1- F1: 5′-CTGTCTTCCAGATACTCGGGATAC-3′
Primer 2- R1: 5′-CCAATGGTGCTTGGACAATGTG-3′
Primer 3- R2: 5′-ATCGCCTTCTATCGCCTTCTTGACGAGTTC-3′

Mitochondrial DNA was quantified by determining the ratios of encoded mitochondrial cytochrome c oxidase (mt-Co2) and cytochrome b (mt-Cybt) to nuclear intron of hemoglobin beta (Hbb) and glucagon (Gcg), respectively, by qRT–PCR of isolated DNA. Total DNA was extracted from liver using the DNeasy Qiagen Kit (Qiagen, Germantown, MD) and amplified using published primer sequences [16 (link)] and quantitative PCR (7900HT Sequence Detection System; Life Technologies, Carlsbad, CA). All primers were synthesized by Sigma-Aldrich (St. Louis, MO, USA).