Versagene rna tissue kit

Total RNA was isolated from mouse brown adipose tissue by using the RNeasy Lipid Tissue Mini Kit. iBAT tissue pieces of 10 mg from 5 mice from each group were pooled for total RNA extraction to represent wild type and Decr^−/− sample. RNA concentration and quality was analyzed using 2100 Bioanalyzer (Agilent Technologies). Total RNA from mouse liver was extracted by using using Versagene RNA tissue kit (Gentra Systems, Minneapolis, MN, USA). The RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) was used to produce cDNA from 1 μg of iBAT RNA or 500 ng of hepatic RNA. The 7500 Real Time PCR System was used with fluorogenic probe-based TaqMan chemistry and the relative standard curve method. TaqMan Gene Expression Assays (Supplementary Table S2) and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were used for iBAT samples throughout the qRT-PCR analyse. For hepatic FGF21, the primers and 5′FAM-labeled probe were designed using Primer Express software (Applied Biosystems) and the sequences available in Genbank and were purchased from Sigma-Genosys (Haverhill, UK). Mouse β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as endogenous controls to which sample values were normalized. Data was analyzed with DataAssist Software v3.0 (Applied Biosystems).

Total RNA was isolated from 1 month old mice (Versagene RNA tissue kit, Gentra Systems), digested with DNaseI and quantitated with ND-1000 spectrophotometer (Nanodrop, Thermo Scientific). Primer sequences were designed in-house with Vector NTI v.10 (Invitrogen). Briefly, 1 μg total RNA was reverse transcribed with Improm-II Reverse Transcriptase (Promega) using random decamers and oligo-dT primers. PCR was performed under standard conditions using 1–2 μl of five-fold diluted first strand cDNA. PCR primers spanned the GARP2-myc junction to distinguish transgenic transcripts from WT. The PCR reactions were repeated three times and analyzed by agarose gel electrophoresis.
Generation of custom GARP polyclonal antibodies- Garp-N antibody was generated in chicken against the most N-terminal 15 amino acids of human GARP protein. The antibodies were purified from the egg yolks using an IgY purification kit and used for immunoblot and immunolocalization. A GARP2 C-terminal specific antibody was generated in rabbit against the last 12 amino acids (Genscript USA, Inc.). Production bleeds were analyzed with ELISA. Serum from rabbit 463 was used in immunoblot and immunolocalization experiments.