Fam labeled taqman probe

Whole epididymal white adipose tissue and liver were pulverized by a homogenizer in RiboZol RNA Extraction Reagent (VWR; Radnor, PA, USA), as instructed by the manufacturer. RNA was quantified using a Nanodrop spectrometer at an absorbance of A260. cDNA was synthesized from 2 μg of RNA using the High Capacity Reverse Transcription Kit (Applied Biosystems; Foster City, CA, USA). The generated cDNA was assessed for gene expression while using FAM-labeled Taqman probes (Applied Biosystems; Foster City, CA, USA). Real time PCR was performed on the 7900HT Fast Real-Time PCR System (Applied Biosystems; Foster City, CA, USA). FAM-labeled Taqman probes were purchased from (Applied Biosystems; Foster City, CA, USA). Threshold cycle values were normalized to the housekeeping gene Gapdh.

After infection, macrophages were washed once with DPBS and mRNA was purified by using the RNeasy purification kit from QIAGEN, according to the manufacturer’s recommendations. For SOCS3 and NOS2 detection, at least 0.8 μg and 1.5 μg of mRNA was then reverse-transcribed using the Reverse Transcription System kit from PROMEGA, respectively. Relative expression of SOCS3 and NOS2 cDNA was detected using specific FAM-labeled Taqman Probe from Life Technologies which targeted human SOCS3 (Hs01000485) or human NOS2 (Hs01075529). We used GAPDH as a housekeeping gene. GAPDH was detected using FAM-labeled Taqman Probe from Life technologies targeting human GAPDH (Hs02758991). FAM fluorescence was detected using LightCycler 480 (ROCHE), and the crossing points (Cp) of PCR reactions were calculated using the Basic Relative Quantification method. Data were then normalized and quantified using the 2^−∆Cp method.

To assess gene expression, peripheral whole blood drawn into Tempus Tubes (Tempus Blood RNA Tube, Life Technologies, Carlsbad, CA) at baseline, 30- and 120-min post-TSST on both study days was stored for up to five days at 4°C, and RNA was then extracted and isolated using Tempus Spin RNA Isolation Kits (Life Technologies, Carlsbad, CA). Aliquots were stored at −80°C until further processing. One-step RT-PCR was performed using Qiagen Quantifast Mastermix kit (Qiagen, Germantown, MD) and commercially available primers (Life Technologies) for IL-6 (00985639_m1), IL-1β (01555410_m1), RelA (00153294_m1), and IκB (00153283_m1) on a RealPlex 4S (Eppendorf, New Brunswick, NJ). The conditions for the RT cycler were 10 min at 50°C, 5 min at 95°C, and 40 cycles of 10 s at 95°C and 30 s at 60°C. Fluorescence data was collected during the extension step of the reaction using 5′ nuclease activity of FAM-labeled TaqMan probes (Life Technologies). Expression of IL-6, IL-1β, IκB, and NF-κB was normalized against expression of endogenous control GAPDH using the ΔΔCt method (ΔΔCt = ΔCt target – ΔCt control), selected because it does not respond to psychosocial stress. For each TSST, gene expression was normalized to each participant’s baseline sample level.