RNA was isolated as previously described. 1 µg RNA was transcribed to cDNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, San Diego, USA). For final measurements 2.5 µl cDNA (1:10), 0.25 µl forward and reverse primer (Biomers, Ulm, Germany), 2.0 µl water and 5.0 µl LightCycler 480 SYBR Green I Master reaction mixture (Roche, Penzberg, Germany) was mixed. After incubation for 5 min at 95°C, gene expression was determined by performing 45 cycles of denaturation (95°C, 10 s), annealing (specific annealing temperature, 15 s) and elongation (72°C, 20 s). Thereafter melting curve analysis was done. Quantitative real-time PCR (qPCR) was performed using LightCycler 480 (Roche, Penzberg, Germany) under conditions described in Table 2 . As IGFBP3 and GDF11 showed aberrant or ambiguous serum concentrations, relative mRNA expression levels were measured and normalized to relative β-actin (β-ACTIN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β2-micro-globulin (β2M) mRNA expression. Results were calculated using delta-delta Ct method considering PCR efficiency. Technical triplicates were performed for each biological sample.
Lightcycler 480 sybr green 1 master reaction mixture
Manufactured by Roche
Sourced in Germany
The LightCycler 480 SYBR Green I Master reaction mixture is a premixed solution containing SYBR Green I dye, DNA polymerase, and necessary reagents for real-time PCR amplification and detection. It is designed for use with the LightCycler 480 Instrument.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480, by Roche (6 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
Forward and reverse primer, by Biomers (5 mentions)
Lightcycler 480 instrument 2 system, by Roche (1 mentions)
Relative human telomere length quantification qpcr assay kit, by ScienCell (1 mentions)
7 protocols using lightcycler 480 sybr green 1 master reaction mixture
1
Quantitative Real-Time PCR Analysis
2
Quantitative Real-Time PCR Analysis
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An amount of 1 µg total RNA was transcribed using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) for complementary DNA (cDNA) synthesis. The qRT-PCR measurements were performed with reaction mixtures containing 5 µL LightCycler 480 SYBR Green I Master reaction mixture (Roche, Basel, Switzerland), 0.25 µL 25 µM forward and reverse primer (Biomers, Ulm, Germany), 2 µL water, and 2.5 µL 1:10 diluted cDNA. Primer sequences are listed in Table 2 . Amplification and detection were accomplished on a LightCycler 480 Instrument II system (Roche, Basel, Switzerland). The PCR program included initial incubation for 5 min at 95 °C, 45 cycles of denaturation (95 °C, 10 s), annealing (specific annealing temperature, 15 s), and elongation (72 °C, 20 s). Following amplification, a melting curve analysis was performed. Relative mRNA gene expression was normalized on the relative succinate dehydrogenase complex flavoprotein subunit A (SDHA), hydroxymethylbilane synthase (HMBS), and ribosomal protein L13 (RPL13) gene expression was calculated based on the delta-delta Ct method considering PCR efficiency and internal calibration. Each condition was measured in biological and technical triplicates.
3
cDNA Synthesis and qPCR Analysis
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A total of 1 µg RNA was transcribed into cDNA by using the SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, San Diego, CA, USA), as described by the manufacturer. Regarding all gene expression measurements, 2.5 µL cDNA (1:10 diluted in water), 0.25 µL forward and reverse primer (Biomers, Ulm, Germany) with a final concentration of 25 µM, 2 µL water and 5 µL LightCycler 480 SYBR Green I Master reaction mixture (Roche, Penzberg, Germany) were used. The protocol for quantitative real-time PCR (qPCR) is shown in Table 2 . The mRNA expression of the genes ß-actin (ACTB), glyceraldehyde-3-phosphatase-dehydrogenase (GAPDH) and ß2-microglobulin (ß2M) were used for normalization. Finally, a melting curve was measured after amplification for the analysis of purity. Three technical replicates were performed for each biological sample. All measurements were carried out by using a LightCycler480 (Roche, Penzberg, Germany). The ΔΔCt method, considering PCR efficiency, was used for the calculation of relative mRNA expression. All sequences of the primer used for qPCR are listed in Table S1 .
4
Quantifying mRNA Levels by qPCR
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For cDNA synthesis, 1 µg RNA was transcribed into cDNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). For p27, p53, p21, LMNB1, IL6, MCP1 and ICAM1 2.5 μL cDNA (1:10), 0.25 μL forward and reverse primer (Biomers, Ulm, Germany), 2.0 μL water and 5.0 μL LightCycler 480 SYBR Green I Master reaction mixture (Roche, Penzberg, Germany) was mixed for each measurement. After an initial incubation for 5 min at 95 °C, the qPCR protocol involves 45 cycles of denaturation (95 °C, 10 s), annealing (specific annealing temperature, 15 s) and elongation (72 °C, 20 s). Relative mRNA expression of measured targets was normalized to relative β-actin (β-ACTIN), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β2-microglobulin (β2M) mRNA expression. After amplification, melting curve analysis was performed. Measurements of gene expression were conducted using LightCycler 480 (Roche, Penzberg, Germany). Relative mRNA expression was calculated using the delta-delta Ct method considering PCR efficiency. Technical triplicates were done for each biological sample. Sequences of forward and reverse primers can be found in Table 3 .
5
Gene Expression Analysis by qPCR
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SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, San Diego, CA, USA) was used to transcribe 1 µg RNA into cDNA. For all gene expression measurements, 2.5 μL cDNA (1:10), 0.25 μL forward and reverse primer (Biomers, Ulm, Germany) with a final concentration of 25 µM (except IL6: 20 µM), 2.0 μL water and 5.0 μL LightCycler 480 SYBR Green I Master reaction mixture (Roche, Penzberg, Germany) were mixed. The qPCR protocol involves an initial incubation for 5 min at 95 °C and 45 subsequent cycles of denaturation (95 °C, 10 s), annealing (specific annealing temperature, 15 s) and elongation (72 °C, 20 s). β-actin (ACTB), glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) and β2-microglobulin (β2M) mRNA expression was used for normalization. Melting curve analysis was done after amplification and technical triplicates were performed for each biological sample. Measurements were carried out using a LightCycler 480 (Roche, Penzberg, Germany). Relative mRNA expression was calculated using the delta delta Ct method considering PCR efficiency. Sequences of the primers used for qPCR are listed in Table 2 .
6
Quantitative RT-PCR Analysis of Gene Expression
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Real-Time quantitative PCR (qPCR) was performed using LightCycler 480 and LightCycler 480 SYBR Green I Master reaction mixture (Roche, Penzberg, Germany). RNA was isolated as described above, whereupon 2 μg were used for cDNA-synthesis (SuperScript II Reverse Transcriptase, Invitrogen- Life technologies, Darmstadt, Germany).
Primer sequences are listed in Additional file2 : Table S2. cDNA was used at 1:10 dilution; targets with low gene expression were measured using 1:5 diluted cDNA. A cutoff for no detectable mRNA expression was set to a Ct level of 35 for further relative gene expression analysis (carried out as described before [18 (link)]), with ACTB, GAPDH and β2M as reference genes, according to MIQE guidelines [39 (link)].
Primer sequences are listed in Additional file
7
Quantifying Relative Telomere Length
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The DNA isolation was done as described previously. The determination of the relative telomere length was performed using the Relative Human Telomere Length Quan-tification qPCR Assay Kit (#8908, ScienCell, Carlsbad, CA, USA). The primer available binds on any specific region on telomeres because they consist of repetitive sequences. In the case of a multiple binding of the primer to telomere, the DNA-polymerase remove those by their specific exonuclease activity. An amount of 2.5 µL DNA (3 ng/µL) was mixed with 10 µL LightCycler 480 SYBR Green I Master reaction mixture (4707516001, Roche, Mannheim, Germany), 5.5 µL water and 2.0 µL of either the telomere or the SCR primer set for the final measurement. Measurements were performed using LightCycler 480 (Roche, Mannheim, Germany). The protocol for this was divided into initial denaturation (10 min, 95 °C), 32 cycles of denaturation (20 s, 95 °C), annealing (20 s 52 °C), elongation (45 s 72 °C) and generation of a melting curve for 15 min (65-97 °C). The SCR primer supplied was used for normalization; it generates an amplificon with a length of 100 bp. The ∆∆ Ct method was used for the calculation of the relative telomere length.
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