Sequence detection system software sds version 2.2

To quantify specific hypothalamic mRNA transcripts, mice were sacrificed at study completion and the hypothalamus rapidly dissected and flash frozen. Briefly, the brain was removed and, using a brain matrix, a 2.0 mm thick coronal slice including the entire hypothalamus was made. Then, a Harris Unicore 1.0 mm tissue punch (Ted Pella, Inc, Redding, CA) was used to dissect the hypothalamus such that the total tissue punch was a cylinder 2.0 mm in length and 1.0 mm in diameter, providing consistency in the amount of RNA isolated. Individual tissue samples were homogenized (Dounce) and RNA was isolated using Qiagen RNeasy Micro Kit (Kit# 74004, Hilden, Germany) and isolated RNA concentrations were quantified by Nanodrop (Thermo Fisher Scientific, Wilmington, DE). qRT-PCR was performed using SYBR Green One-Step (Kit# 600825, Agilent, Santa Clara, CA). qRT-PCR data were analyzed using the Sequence Detection System software (SDS Version 2.2; Applied Biosystems, Foster City, CA). Expression levels of each gene were normalized to a housekeeping gene (18S RNA) and standard curve. Non-template controls were incorporated into each PCR run. Selected oligonucleotides are found in the Key Resources Table.

Total RNA was extracted from BAT using TRIzol according to manufacturer's instructions (MRC, Cincinnati, OH). RNA was quantitated by spectrophotometry at 260 nm (Nanodrop 1000; Thermo Scientific, DE) and reverse-transcribed with AMV reverse transcriptase (1 μg) (Promega, Madison, WI), and real-time PCR was performed on a ABI Prism 7900 HT (Applied Biosystems) using the commercially available PCR master mix (SYBR Green, Applied 2.0; Applied Biosystems), as described previously [20] (link). PCR data were analyzed using the Sequence Detection System software (SDS Version 2.2; Applied Biosystems). Expression levels of each gene were normalized to a housekeeping gene (18S RNA) and expressed as a percentage of controls. Non template controls were incorporated into each PCR run.