Rnaeasy midi kit

Male and female mice were injected with dexamethasone (5 mg/kg; i.p.). Three hours later amygdala and NAc punches were collected for qPCR analysis. Total cellular RNA was extracted from amygdala and NAc punches using Qiazol reagent and the RNAeasy Midi kit (QIAGEN, Valencia, CA, United States). Total RNA was reverse transcribed into cDNA using VILO master mix (Invitrogen, Carlsbad, CA, United States). qPCR was performed in triplicate aliquots from each individual animal with Power SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, United States), 25 ng of cDNA and 0.5 μM of primers using an ABI Prism 7900HT (Thermo Fisher Scientific, Waltham, MA, United States) in the qPCR CoRE at Icahn School of Medicine at Mount Sinai. Primer sequences for GPR83, proSAAS and GAPDH are the same as used previously (Fakira et al., 2019 (link)). The primer sequences used for qPCR are: GAPDH Forward: 5′-TGAAGGTCGGTGTGAACG Reverse: 5′-CAATCTCCACTTTGCCACTG, GPR83 Forward: 5′-GCAGTGAGATGCTTGGGTTC Reverse: 5′-CCCACCAAT AGTATGGCTCA, and proSAAS: Forward: 5′-AGTGTATG ATGATGGCCC Reverse: 5′-CCCTAGCAAGTACCTCAG. The CT values for the technical replicates were averaged and analysis performed using the ΔΔC_T method and normalized to saline controls. In some cases, qPCR reactions were repeated to determine the reliability of the primers and RNA samples.

RNA was isolated from 100–150 mg subcutaneous or omental human fat tissue and 20 mg of the corresponding liver biopsy using the RNA Easy Midi kit (Qiagen). For the human fetal and adult livers, RNA was isolated as indicated above. cDNA was synthesized from 0.1 μg RNA using SuperScript® III First-Strand Synthesis SuperMix (Invitrogen) with Oligo(dT) primers. cDNA was subjected to quantitative real time PCR (qRT-PCR) analysis to quantify mRNA levels of mARC1 and mARC2 using the TaqMan Gene Expression Assays Hs00224227_m1 and Hs00215486_m1, respectively (Invitrogen). As a housekeeping gene the TATA box binding protein (TBP) was used for normalization. The relative expression levels were defined by the 2^-ΔΔCt method as described elsewhere [10 (link)].

After euthanasia, before perfusion, a sample of white matter from the opposite hemisphere to that used for immunohistochemistry was obtained and placed in RNAlater solution (Qiagen, West Sussex, UK), frozen in liquid nitrogen and stored at −80 ℃ until processing. RNA was extracted using the standard protocol for animal tissues supplied with the RNAeasy Midi kit (Qiagen, West Sussex, UK). RNA quality was assessed using a Nanodrop spectrophotometer (NanoDrop, Wilmington, DE, USA) and Agilent 2100 Bioanalyser (Agilent, Santa Clara, CA, USA) and all samples had a spectral 260/280 ratio of between 2.05 and 2.13, and a RNA Integrity number of 9.9–10.