Taqman assays with amplitaq gold dna polymerase

Total RNA was obtained from frozen mouse white adipose tissue via mechanical homogenization at 4.5 m/sec for 30 sec using a FastPrep‐24 tissue homogenizer (MP Biomedicals, Santa Ana, CA, USA) and ceramic beads, followed by guanidinium thiocyanate–phenol–chloroform extraction. RNA was treated with DNase I (Cat# 18068‐015; Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was prepared using 1000 ng total RNA and SuperScript III Reverse Transcriptase (Cat# 18080‐044; Thermo Fisher Scientific, Waltham, MA, USA). Transcript expression was measured using TaqMan Assays with AmpliTaq Gold DNA polymerase (Cat# N8080247; Thermo Fisher Scientific, Waltham, MA, USA) in a Rotor‐Gene Q real‐time PCR cycler (QIAGEN, Hilden, Germany), and target genes were compared to the geometric mean of Rplp0 and Rn18s housekeeping genes using the ΔΔC_T method. Gene expression was analyzed in WT mice treated with vehicle (n = 7), WT mice treated with fucoidan (n = 5), SRA^−/− mice treated with vehicle (n = 6) and SRA^−/− mice treated with fucoidan (n = 8).

Total RNA was obtained from ∼50 mg of epidydimal WAT or liver via mechanical homogenization at 4.5 m/s for 45 s using a FastPrep‐24 tissue homogenizer (MP Biomedicals) and glass beads, followed by phenol‐chloroform extraction. RNA was treated with DNase I (Thermo Fisher Scientific) and cDNA was prepared using 500–1000 ng total RNA and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Transcript expression was measured using TaqMan Assays with AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific) and target genes were compared to the mean of Rplp0 and 18S housekeeping genes using the ΔΔC_T method.