Fast probe qpcr master mix

In the experiment, the relative expression of the Tnfrsf1a and Cav1 genes was measured, using Hprt and Tbp as endogenous controls (Table 1). The reaction was performed in triplicate using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Fast Probe qPCR Master Mix (2x) plus ROX solution (EURx, Gdańsk, Poland). The reaction mixture contained 10 µL of Fast Probe qPCR Master Mix (2x), 9 µL of RNase-free water, ROX solution (50 nM), and 0.5 µM of the gene-specific TaqMan probe (Applied Biosystems, Foster City, CA, USA). Data quality screening was performed based on amplification, Tm and Ct values to remove any outliers before calculating ΔΔCt and determining the fold change in mRNA levels. The data is presented as an RQ value (RQ = 2 − ΔΔCt).
The primer sequences, gene symbols, assay IDs, gene names, and amplicon lengths (bp) for the genes analyzed are presented in Table 1.

The relative expression of the following genes was measured: Slc2a3, Gapdh, Ldha, Ldhb, and Pkfb3 by real-time PCR, ΔΔCt, using Hprt and Tbp as endogenous controls (Table 1). The reaction was performed in successive triplicates using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Fast Probe qPCR Master Mix (2×) plus ROX solution (EURx, Gdańsk, Poland). The reaction mixture contained 10 µL of Fast Probe qPCR Master Mix (2×), 9 µL of RNase-free water, ROX solution (50 nM), and 0.5 µM of the gene-specific TaqMan probe (Applied Biosystems, Foster City, CA, USA). Data quality screening was performed based on amplification, Tm, and Ct values to remove any outliers before calculating ΔΔCt, and determining the fold change in mRNA levels. Data are presented as RQ value (RQ = 2 − ΔΔCt).

All samples of good quality (OD 260/280 ratios approximately 2.0) were reverse transcribed using random primers and NG dART RT-PCR reagents (EURx, Gdańsk, Poland) as described by the manufacturer. To analyze the correlation of diabetes with the cognitive function, the following primers were used: Arc, Bdnf, Egr1 [see Table 1]. The analysis of the genes’ expression levels was performed by real-time PCR method using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Fast Probe qPCR Master Mix (2x), plus ROX Solution (EURx, Poland). Briefly, the reaction mixture contained 10 µL of Fast Probe qPCR Master Mix (2x), 9 µL of RNase-free water, ROX Solution (50 nM), and 0.5 µM of gene-specific TaqMan probe (Applied Biosystems, Foster City, CA, USA). The reactions were performed as followed: 95 °C for 3 min, 40 cycles: 95 °C for 10 s and 60 °C for 30 s. The data quality screen based on amplification, Tm and Ct values was performed to remove any outlier data before ΔΔCt calculations and to determine fold change in mRNA levels. The data were presented as a mean RQ ± SEM value (RQ = 2−ΔΔCt).