Quantifast sybrvr green rt pcr kit

Total RNA was extracted from whole‐brain tissues using TRIzol (Invitrogen, Paisley, UK) according to the manufacturer's instructions. DNAse‐treated RNA samples were analyzed by quantitative RT‐PCR using QuantiFast™ SYBRVR green RT‐PCR kit (Qiagen Inc.) according to the manufacturer's instructions. All reactions were performed using a LightCycler (Roche, Mannheim, Germany). At the end of each PCR run, melting curve analysis was performed to verify the integrity and homogeneity of PCR products. Gene expression levels were calculated using standard curves for each gene, which were created by plotting threshold cycle (CT) values versus the logarithm of serial‐diluted RNA concentrations. A least‐square method was used for the determination of A and B values in the equation CT = A*Log (C_RNA) + B. The coefficient of determination (R²) was greater than 0.99. Values were normalized using the respective values for the housekeeping gene, Gapdh. All results were analyzed using the LightCycler software version 3.5 (Roche, Mannheim, Germany, RRID: rid_000088). QuantiTect Primer Assays were used for Il6 (Mm_Il6_1_SG), Il1b (Mm_Il1b_2_SG), tlr2 (Mm_Tlr2_1_SG), Csf1 (Mm_Csf1_2_SG), Lgals3 (Mm_Lgals3_1_SG), P2ry12 (Mm_P2ry12_3_SG), Nos1 (Mm_Nos1_2_SG), and Gapdh (Mm_Gapdh_3_SG), all QuantiTect Primer Assays from Qiagen.

Total RNA was extracted from ice-cold PBS-perfused, whole-brain tissues using TRIzol (Invitrogen, Paisley, UK) according to the manufacturer's instructions. DNAse-treated RNA samples were analyzed by quantitative RT-PCR using QuantiFast™ SYBRVR green RT PCR kit (Qiagen Inc.) according to the manufacturer's instructions. All reactions were performed using a LightCycler (Roche, Mannheim, Germany). At the end of each PCR run, melting curve analysis was performed to verify the integrity and homogeneity of PCR products. Gene expression levels were calculated using standard curves for each gene, which were created by plotting threshold cycle (CT) values versus the logarithm of serial diluted RNA concentrations. A least-square method was used for the determination of A and B values in the equation CT = A*Log (CRNA) + B. The coefficient of determination (R2) was greater than 0.99. Values were normalized using the respective values for the housekeeping gene, Gapdh. All results were analyzed using the LightCycler software version 3.5 (Roche, Mannheim, Germany, RRID: rid_000088). QuantiTect Primer Assays were used for iNOS (Mm_Nos1_2_SG), Il1b (Mm_Il1b_2_SG), Ym-1 (Mm_Chi3l3_1_SG), Arg1 (Mm_Arg1_1_SG), Olig2 (Mm_Olig2_1_SG), Snap25 (Mm_Snap25_2_DG) and Gapdh (Mm_Gapdh_3_SG), all QuantiTect Primer Assays from Qiagen.