Be the optimal reference genes for normalization.Quantitative Real-time PCRGene expression was quantified on the Mx3000PH and Mx3005TM real-time PCR systems (Stratagene). Primers and dual-labelled hydrolysis probes for the genes of interest and references genes were designed using Beacon DesignerTM (PREMIER Biosoft, USA). The design included a BLAST search for test of sequence homology, a test for secondary structures and optimization of multiplex setup. The genes, accession number, primers, probes and amplicon lengths are listed in Table 2. All primers and probes were purchased from Sigma-Aldrich (SigmaAldrich Danmark A/S, Denmark). For each gene, the optimal primer and probe concentration was established. All samples were run in duplicates for genes of interest and reference genes using 1 ml of cDNA. The Brilliant Multiplex QPCR Master Mix (CAL-120 biological activity Stratagene, cat.no. 600553) was used. The thermal profile employed was 10 minutes of denaturation at 95uC followed by 40 cycles with denaturation for 15 s at 95uC and annealing/elongation at 60uC for 1 minute. The quantification and normalization of results were based on the computation of target quantification cycle (Cq) values and reference gene Cq values in the qbasePLUS software (Biogazelle NV, Belgium). The method of qbasePLUS is a modification of the classic delta-delta-Ct method [16]. Instead of just normalizing to a single reference gene, multiple reference genes are taken into account and correction for gene specific amplification efficiencies can be done. The three reference genes, ACTB, B2M and GUSB, were included in the analysis. The reference target stability was 0.63 (M-value) and 0.27 (CV-value). This is a little higher than what is expected from homogeneous sample panels, however, as these samples include various cell types they are classified as heterogeneous and higher M- and C-values are expected. One default amplification efficiency (100 ) for all targets was used, as the amplification efficiency varied little over the multiple runs. The data were reported as calibrated normalized relative quantities (CNRQs). As all the samples could not be included in a single run, inter-run calibration was done to correct for run-to-run differences. This was based on three inter-run calibrators included in all runs.Gamma 24272870 CountingThe tubes with the aortas were placed in the 2480 Automatic Gamma Counter, Wizard2TM3” (Perkin Elmer, USA). The samples were counted for 120 seconds using a designated 18Fprotocol. The counting efficiency of the gamma counter was tested to be 0.54. During subsequent analysis, SUVs were calculated.RNA Extraction and Reverse TranscriptionTotal RNA was isolated with TRI ReagentH in accordance with the protocol of the manufacturer (Molecular Research Center Inc., USA). The quality of the isolated RNA was tested using the Agilent 2100 Bioanalyzer in conjunction with the Agilent RNA 6000 Nano Kits (Agilent Technologies Denmark A/S, Denmark). A RNA integrity number (RIN)-value above 5 was 223488-57-1 web accepted [14]. The quantity of RNA was measured using the NanoDrop 1000 (Thermo Fischer Scientific, USA). Total RNA (0.3 mg) was reversed transcribed (RT) using the AffinityScriptTM QPCR cDNA Synthesis Kit (Stratagene, USA, cat.no. 600559) according to the protocol of the manufacturer. The RT was performed using 1662274 the Eppendorf Mastercycler Gradient (Eppendorf AG, Germany) with the following protocol: incubation at 25uC for 5 minutes (primer annealing), 42uC for 15 minutes (cDNA synthesis) a.Be the optimal reference genes for normalization.Quantitative Real-time PCRGene expression was quantified on the Mx3000PH and Mx3005TM real-time PCR systems (Stratagene). Primers and dual-labelled hydrolysis probes for the genes of interest and references genes were designed using Beacon DesignerTM (PREMIER Biosoft, USA). The design included a BLAST search for test of sequence homology, a test for secondary structures and optimization of multiplex setup. The genes, accession number, primers, probes and amplicon lengths are listed in Table 2. All primers and probes were purchased from Sigma-Aldrich (SigmaAldrich Danmark A/S, Denmark). For each gene, the optimal primer and probe concentration was established. All samples were run in duplicates for genes of interest and reference genes using 1 ml of cDNA. The Brilliant Multiplex QPCR Master Mix (Stratagene, cat.no. 600553) was used. The thermal profile employed was 10 minutes of denaturation at 95uC followed by 40 cycles with denaturation for 15 s at 95uC and annealing/elongation at 60uC for 1 minute. The quantification and normalization of results were based on the computation of target quantification cycle (Cq) values and reference gene Cq values in the qbasePLUS software (Biogazelle NV, Belgium). The method of qbasePLUS is a modification of the classic delta-delta-Ct method [16]. Instead of just normalizing to a single reference gene, multiple reference genes are taken into account and correction for gene specific amplification efficiencies can be done. The three reference genes, ACTB, B2M and GUSB, were included in the analysis. The reference target stability was 0.63 (M-value) and 0.27 (CV-value). This is a little higher than what is expected from homogeneous sample panels, however, as these samples include various cell types they are classified as heterogeneous and higher M- and C-values are expected. One default amplification efficiency (100 ) for all targets was used, as the amplification efficiency varied little over the multiple runs. The data were reported as calibrated normalized relative quantities (CNRQs). As all the samples could not be included in a single run, inter-run calibration was done to correct for run-to-run differences. This was based on three inter-run calibrators included in all runs.Gamma 24272870 CountingThe tubes with the aortas were placed in the 2480 Automatic Gamma Counter, Wizard2TM3” (Perkin Elmer, USA). The samples were counted for 120 seconds using a designated 18Fprotocol. The counting efficiency of the gamma counter was tested to be 0.54. During subsequent analysis, SUVs were calculated.RNA Extraction and Reverse TranscriptionTotal RNA was isolated with TRI ReagentH in accordance with the protocol of the manufacturer (Molecular Research Center Inc., USA). The quality of the isolated RNA was tested using the Agilent 2100 Bioanalyzer in conjunction with the Agilent RNA 6000 Nano Kits (Agilent Technologies Denmark A/S, Denmark). A RNA integrity number (RIN)-value above 5 was accepted [14]. The quantity of RNA was measured using the NanoDrop 1000 (Thermo Fischer Scientific, USA). Total RNA (0.3 mg) was reversed transcribed (RT) using the AffinityScriptTM QPCR cDNA Synthesis Kit (Stratagene, USA, cat.no. 600559) according to the protocol of the manufacturer. The RT was performed using 1662274 the Eppendorf Mastercycler Gradient (Eppendorf AG, Germany) with the following protocol: incubation at 25uC for 5 minutes (primer annealing), 42uC for 15 minutes (cDNA synthesis) a.
