7500 fast real time pcr system software

Quantitative mRNA expression of PTGS1 (NCBI Reference Sequence: NM_000962) and PTGS2 (NM_000963) genes was performed in a two-step reverse transcription PCR. Total RNA was extracted from cells using RiboPure kit (Life Technologies, USA). After determination of the quantity and quality of isolated RNA using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA), complementary DNA (cDNA) was prepared using RevertAid First Strand cDNA Synthesis kit (Fermentas, Thermo Scientiic, Lithuania). Quantitative real-time PCR was performed in 7500 Fast Real-Time PCR System (Applied Biosystems, USA), using pre-validated Taqman Gene Expression Assays TaqMan GE Master Mix (Applied Biosystems, USA) and 1.5 μl of cDNA for each reaction mix of 15 μl. Each sample was analyzed in two technical replicates, and mean C_T values were used for further analysis. Calculations were performed using the ΔΔCt relative quantification method, using 7500 Fast Real-Time PCR System Software (Applied Biosystems, USA). The thresholds were set manually to compare data between runs, and C_T values were extracted. All C_T values for each sample were normalized to the value obtained for GAPDH and HPRT1, the endogenous control gene. Fold change between groups was calculated from the means of the logarithmic expression values.

Total RNA was extracted using RNeasy mini extraction kit (Qiagen, Valencia, CA, USA) for cultured peritoneal macrophages or Tri reagent (MRC, OH, USA) for brain tissues. Total RNA was reverse-transcribed using oligo (dT) primers and the SuperScript First-Strand Synthesis System (Invitrogen) according to the manufacturer’s protocol. PCR primers and probes specific for MCP-1, IL-6, CCR2, CD36, and β-actin (an internal control) were obtained as TaqMan pre-developed optimized assay reagents for gene expression (Applied Biosystems, Foster City, CA, USA). The PCR reaction was performed using TaqMan Universal PCR Mastermix, No AmpErase UNG, and 7500 Fast Real-Time PCR system (Applied Biosystems) according to the manufacturer’s protocol. Reactions were performed in 20 μL total volume and incubated at 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C, and 1 min at 60°C. The results were analyzed by 7500 Fast Real-Time PCR system software (Applied Biosystems).

The mice were killed by asphyxiation with CO₂ and placed in a stereotaxic frame. The atlanto-occipital membrane was exposed, and cerebrospinal fluid was withdrawn from the cisterna magna using a Hamilton syringe mounted on a micromanipulator and immediately frozen. A hypothalamic block was then dissected and placed in RNAlater stabilization reagent (Qiagen) and stored at −70°C until analysis.
The concentration of PGE₂ in the cerebrospinal fluid (diluted 1:100) was determined using a High Sensitivity Prostaglandin E2 Enzyme Immunoassay Kit (Assay Designs). The values were calculated using a standard curve ranging from 7.81 to 1000 pg/ml (R² = 1).
RNA was extracted from the hypothalamus with RNeasy Plus Universal Kit (Qiagen), and reverse transcription was done with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems); qPCR was performed using Gene Expression Master Mix (Applied Biosystems) on a 96-well plate (7500 Fast Real-Time PCR System Software; Applied Biosystems). Mm00477214_m1 (Cox-1), Mm00478374_m1 (Cox-2), Mm00452105_m1 (mPGES-1), and Mm99999915_g1 (GAPDH) TaqMan assays (Applied Biosystems) were used.

Quantitative expression of mRNA of PTGS1 (National Center for Biotechnology Information (NCBI) Reference Sequence: NM_000962) and PTGS2 (NM_000963) genes was performed in a two-stage reverse transcription PCR. Total RNA was isolated from cells with an AllPrep DNA/RNA Mini Kit. Concentrations and purity of obtained RNA were measured by the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Waltham, MA, USA). The RNA was reverse-transcribed using a high capacity cDNA Reverse Transcription Kit with random primers, according to the manufacturer’s instructions. Quantitative assessment of mRNA levels was performed by the 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA), with Power SYBR Green PCR Master Mix reagent. Every sample was analyzed simultaneously in two technical replicates and mean cycle threshold (CT) values were used for further analysis. The relative quantification method was applied in calculations, using 7500 Fast Real-Time PCR System Software (Applied Biosystems, Foster City, CA, USA). The relative quantity of a target, normalized to the endogenous control GAPDH gene. Analysis of these relative changes in gene expression between samples was performed using the 2^−ΔΔCT method [65 (link)].