Taqman universal master mix with uracil n glycosylase

Real-time qPCR was performed in triplicate using QuantStudio 6 (Applied Biosystems) and TaqMan universal master mix with uracil-N-glycosylase (Applied Biosystems, Waltham, MA). Reactions contained 1× master mix, 250 ng of RNA, and 1× dilution of each primer-probe set in a total volume of 20 μL (50°C for 2 minutes, 95°C for 15 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 60 seconds). Taqman primer-probe pairs were purchased from Applied Biosystems: IL-10 (Hs00961622_m1), IL-12b (Hs01011518_m1), ARG1 (Hs00968979_m1), NOS2 (Hs01075529_m1), or GAPDH (Hs02758991_g1).

For rat brain lysates, total RNA was converted to cDNA using random primers and
Life Technologies Superscript II. The cDNA was diluted (5 ng per µl) and Taqman
reactions performed with 20 ng cDNA per well under standard conditions in 25 µl
reaction volume. Taqman probesets for rat Sgk1 and 18S ribosomal RNA were
obtained from Applied Biosystems, Paisley, UK (Rn00570285_m1 and #4310893E,
respectively). For mouse brain lysates, total RNA was isolated from cerebra of
young adult male mice from the Sgk1^+/+ and Sgk1^−/−colonies. Whole cerebral hemispheres were homogenized and passed through
QIAshredder™ columns (QIAGEN GmbH, Hilden, Germany). RNA was extracted using an
RNeasy® Mini kit (QIAGEN GmbH, Hilden, Germany). Total RNA (500 ng) was
converted to cDNA using a Precision Nanoscript reverse transcription kit with
random primers (Primerdesign Ltd, Southampton, UK). Amplification reactions were
performed in a 20-µl reaction volume containing 250 ng cDNA for Sgk3 and Sgk1
assays, and 25 ng cDNA for the housekeeping gene Gapdh, using Taqman® Universal
mastermix with uracil-N-glycosylase (Applied Biosystems, Paisley, UK). Taqman®
expression assays for mouse Sgk3 (assay # Mm01227735_m1), Sgk1 (Mm00441387_g1)
and gapdh (Mm99999915_g1) were purchased from Applied Biosystems-Life
Technologies, Paisley, UK.