Taqman rt pcr reagents

IM tissue was collected and stored in RNAlater solution (Life Technologies) until homogenization. Purified RNA was extracted using PureLink RNA Mini Kit (Ambion ‐ Life Technologies), and quantified via UV spectrophotometry. Taqman RT‐PCR reagents (Applied Biosystems) were used to generate all cDNA. Using predeveloped Taqman assays, real‐time quantitative PCR was performed for P2Y₂ and the housekeeping transcript 18S (Applied Biosystems, Carlsbad, CA). PCR was performed with 20 ng of cDNA and the corresponding primer pairs using an iCycler Real‐Time Detection System (BioRad Laboratories). The relative quantity of mRNA was determined using the comparative Ct method and expression levels of P2Y₂ receptor mRNA were normalized to ribosomal 18S expression.

The real-time RT-PCR analysis was conducted using the ABI7900HT machine (Applied Biosystems). All of the TaqMan RT-PCR reagents, including the primers and probes, were purchased from Applied Biosystems. The TaqMan analysis was conducted using predesigned and optimized Assays on Demand (Applied Biosystems). The following assays were used: HIF-1α (ID: Hs00936371_m1), HIF-2α (ID: Hs01026149_m1), OCT4 (ID: Hs03005111_g1), NANOG (ID: Hs02387400_g1), SOX2 (ID: Hs01053049_s1), REX1 (ID: Hs01938187_s1), MIF (ID: Hs00236988_g1), KLF2 (Hs00360439_g1), PPAR γ (ID: Hs01115729_m1), and GAPDH (ID: Hs00266705_g1). The reaction parameters are 2 min at 50 °C hold, 30 min at 60 °C hold, and 5 min at 95 °C hold, and this was followed by 45 cycles of 20 s at 94 °C for a melting, and 1 min at 60 °C for an annealing/extension. All of the measurements were performed in duplicate or triplicate. The results were analyzed using the ABI sequence-detection software version 2.0 (Applied Biosystems). A relative quantitation was conducted using the GAPDH as a reference gene, and it was validated using the NormFinder software. Since all of the used assays were optimized for PCR efficiency by the manufacturer, the mRNA-expression levels were estimated according to the delta-Ct values.

Real-time RT-PCR analysis was conducted using an ABI7900HT machine (Applied Biosystems). All TaqMan RT-PCR reagents, including primers and probes, were purchased from Applied Biosystems. TaqMan analysis was conducted using predesigned and optimized Assays on Demand (Applied Biosystems). The following assays were used: HIF-1α (ID: Hs00936371_m1), HIF-2α (ID: Hs01026149_m1), OCT4 (ID: Hs03005111_g1), NANOG (ID: Hs02387400_g1), SOX2 (ID: Hs01053049_s1), REX1 (ID: Hs01938187_s1), GAPDH (ID: Hs00266705_g1). The reaction parameters were as follows: 2-min 50°C hold, 30-min 60°C hold, and 5-min 95°C hold, followed by 45 cycles of 20-s 94°C melting and 1-min 60°C annealing/extension. All measurements were performed in duplicate or triplicate and the results were analyzed using the ABI sequence detector software, version 2.0 (Applied Biosystems). Relative quantitation was conducted using GAPDH as a reference gene, which was validated using the NormFinder software (supporting information in S1 Fig and S1 Table). Since all assays used were optimized for PCR efficiency by the manufacturer, mRNA expression levels were estimated by the delta ct values.

RNA was isolated from the brainstem using the RNeasy Lipid Tissue Mini Kit (Qiagen, Germany) according to manufacturer instructions using the Qiazol Lysis Reagent followed by DNase treatment. RNA samples were quantified by spectrophotometry (Nanodrop ND_100) and subjected to reverse transcription (RT)-PCR (25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min) using the TaqMan RT-PCR Reagents and a GeneAmp PCR System (Applied Biosystems, Singapore) (end volume of 40 μL). The expression levels of genes encoding Cx47, Cx43 and BiP were assessed by quantitative Real-Time PCR Analysis (hold at 55 °C for 2 min and at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min) using a 7900HT Real-Time PCR System (Applied Biosystems) and Taq-Man^© Gene Expression Assays: Cx43: Mm01179639_s1; Cx47 Mm00519131_s1; BIP: Mm00517691_m1, Tubulin (Mm00726185_s1) was used as endogenous “house- keeping” control gene. Each sample was loaded in triplicate and contained 250 ng of cDNA, 1 μl of TaqMan Gene Expression Assay, and 10 μl of TaqMan Gene Expression Master Mix (end volume 20 μl). Expression levels in LPS and saline control mice were calculated after normalizing cycle thresholds against tubulin and presented as the fold induction value (2^{–D DCt}) relative to naive control mice (mean ± standard deviation).