Lightcycler 480 software v1

Manufactured by Roche

The Lightcycler 480 Software v1.5 is a software package designed for use with the Lightcycler 480 real-time PCR instrument. The software provides tools for data acquisition, analysis, and report generation during real-time PCR experiments.

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Lab products found in correlation

Tri reagent, by Merck Group (3 mentions) Isolate 2 rna mini kit, by Meridian Bioscience (2 mentions) Tetro cdna synthesis kit, by Meridian Bioscience (2 mentions) Sensimix sybr low rox kit, by Meridian Bioscience (2 mentions) Lightcycler 480 384 well plate, by Roche (2 mentions) Superscript 3, by Thermo Fisher Scientific (2 mentions) Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Rq1 rnase free dnase kit, by Promega (1 mentions) Lightcycler 480 quantitative pcr system, by Roche (1 mentions) Light cycler 480 multiwell plates, by Roche (1 mentions) Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions) Lightcycler 480 system, by Roche (1 mentions) Agilent bioanalyzer, by Agilent Technologies (1 mentions) Lightcycler 480 sybr green 1 master, by Roche (1 mentions) Ambion dnase 1, by Thermo Fisher Scientific (1 mentions) Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions) Dnase 1, by Thermo Fisher Scientific (1 mentions) Universal probe library, by Roche (1 mentions) Lightcycler 480 instrument, by Roche (1 mentions) Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)

Spelling variants of this product

LightCycler 480 (7708 citations) LightCycler (1119 citations) LightCycler software (129 citations)

7 protocols using lightcycler 480 software v1

Quantifying P2X7R Expression by Real-Time PCR

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The spinal cords were homogenized in TRI reagent (Sigma-Aldrich), and the total RNA was extracted. After treatment with the RQ1 RNase-Free DNase kit to remove contaminating DNA (Promega, Madison, WI), 5 μg of total RNA was reverse transcribed for 1 hour at 50°C using 200 units of SuperScript III reverse transcriptase (Invitrogen). Then, 200 ng of cDNA was used as the template for real-time PCR.
The quantitative PCR reactions were performed in triplicate using a Roche LightCycler 480 quantitative PCR system (Roche Applied Science, Indianapolis, IN). The P2X7R expression levels were normalized to those of GAPDH. Quantitative PCR amplification was performed as 20 μL reactions containing 10 μL of X2 SYBR Green I Master Mix (Roche Applied Science), 0.8 μL of the forward and reverse primers (μM each), and 9.2 μL of the sample or nuclease-free water. All reactions were performed in 96-well plates (LightCycler 480 Multiwell Plates; Roche Applied Science).
The cycling conditions consisted of a 10-minute polymerase activation step at 95°C, 45 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 25 seconds, and a dissociation curve analysis step from 60° to 95°C. The relative quantification results for both P2X7R and GAPDH were calculated according to the second derivative maximum method using LightCycler 480 Software v.1.5 (Roche Applied Science).

Liu P.Y., Lee I.H., Tan P.H., Wang Y.P., Tsai C.F., Lin H.C., Lee F.Y, & Lu C.L. (2015). P2X7 Receptor Mediates Spinal Microglia Activation of Visceral Hyperalgesia in a Rat Model of Chronic Pancreatitis. Cellular and Molecular Gastroenterology and Hepatology, 1(6), 710-720.e5.

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Quantitative Gene Expression Analysis

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In order to determine gene expression levels, RNA was extracted from cultured cells using ISOLATE II RNA Mini Kit (Bioline) and used as a template to generate cDNA with Tetro cDNA Synthesis Kit (Bioline). Quantitative RT-PCR was performed using SensiMix SYBR Low-ROX Kit (Bioline; annealing temperature—60 °C) in a Lightcycler 480 384-well plate (Roche), and analyzed using Lightcycler 480 Software v1.5 (Roche). Mouse Hprt was used as a housekeeping gene. The primer sequences used in this study are listed in the Resources Table (Table 1).

Andronikou C., Burdova K., Dibitetto D., Lieftink C., Malzer E., Kuiken H.J., Gogola E., Ray Chaudhuri A., Beijersbergen R.L., Hanzlikova H., Jonkers J, & Rottenberg S. (2024). PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair. The EMBO Journal, 43(6), 1015-1042.

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Organellar Gene Expression Profiling

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Purified total or mitochondrial RNA was subjected to cDNA synthesis. Reverse transcription was performed with random hexamer primers using Superscript III (Life Technologies) according to the manufacturer’s instructions. Specific primers (supplementary table S1, Supplementary Material online) were designed for representative genes from each organellar DNA using the Primer3 software (version 0.4.0) (http://bioinfo.ut.ee/primer3-0.4.0/primer3/input.htm, last accessed January 15, 2014). Quantitative polymerase chain reaction (qPCR) was performed using Power SYBR Green PCR Master Mix (Life Technologies) on a microwell plate-based cycler platform (LightCycler 480 System, Roche) and the data were analyzed using the LightCycler 480 software v. 1.5 (Roche). Each experiment had three technical replicates. Specificity of the amplification reactions was confirmed by electrophoresis in a 1% agarose gel.

Dobáková E., Flegontov P., Skalický T, & Lukeš J. (2015). Unexpectedly Streamlined Mitochondrial Genome of the Euglenozoan Euglena gracilis. Genome Biology and Evolution, 7(12), 3358-3367.

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Quantifying Gene Expression Levels

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In order to determine gene expression levels, RNA was extracted from cultured cells using ISOLATE II RNA Mini Kit (Bioline) and used as a template to generate cDNA with Tetro cDNA Synthesis Kit (Bioline). Quantitative RT-PCR was performed using SensiMix SYBR Low-ROX Kit (Bioline; annealing temperature −60°C) in a Lightcycler 480 384-well plate (Roche), and analyzed using Lightcycler 480 Software v1.5 (Roche). Mouse Rps20 and human HPRT were used as house-keeping genes. The primer sequences used in this study are listed in Table S2.

Dias M.P., Tripathi V., van der Heijden I., Cong K., Manolika E.M., Bhin J., Gogola E., Galanos P., Annunziato S., Lieftink C., Andújar-Sánchez M., Chakrabarty S., Smith G.C., van de Ven M., Beijersbergen R.L., Bartkova J., Rottenberg S., Cantor S., Bartek J., Chaudhuri A.R, & Jonkers J. (2021). Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps. Molecular cell, 81(22), 4692-4708.e9.

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Quantitative RT-PCR Gene Expression Analysis

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Total RNA was isolated using the Tri Reagent (Sigma) and treated with Ambion^TM Dnase I (Invitrogen) following the manufacturers’ instructions. RNA quality was tested using an Agilent Bioanalyzer. 2μg of total RNA was denatured at 65 °C for 10 min and incubated for 1 h at 50 °C in the presence of 2.5 mM dNTP, 100U Superscript III (Invitrogen) using 0.5μg oligo(dT)15 primer in a total volume of 20 μl, followed by inactivation at 70 °C. A control PCR reaction of the reverse transcription was performed with human GAPDH forward (gaacatcatccctgcatcc) and reverse (ccagtgagcttcccgttca) primers with Q5 High fidelity DNA polymerase according to the manufacturer’s instructions (New England BioLabs^®). Real-time PCR was carried out with The LightCycler^® 480 SYBR Green I Master (Roche Life Science) in triplicates and analyzed using LightCycler® 480 Software, v1.5 (Roche). The expression of each gene was normalized to the HPRT1 or GAPDH housekeeping genes and relative levels were calculated on the basis of the comparative cycle threshold Ct method (2−ΔΔCt) where ΔΔCt is the difference in Ct between target and reference gene. For the list and sequence of RT-qPCR oligonucleotides refer to Supplementary Table 4.

Fayad R., Rojas M.V., Partisani M., Finetti P., Dib S., Abelanet S., Virolle V., Farina A., Cabaud O., Lopez M., Birnbaum D., Bertucci F., Franco M, & Luton F. (2021). EFA6B regulates a stop signal for collective invasion in breast cancer. Nature Communications, 12, 2198.

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RNA Extraction and Real-Time RT-PCR Analysis

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Total RNA was isolated from peritoneal or spleen cells by means of TriReagent^® according to the manufacturer’s instructions (Sigma Aldrich). After quantifying by absorbance at 260 nm/280 nm, DNA contamination was degraded by DNaseI (Fermentas, St. Leon Rot, France). The single-strand cDNA synthesis was performed using a Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics). Optimal primer design and probe selection for mRNA detection of selected genes from the Universal ProbeLibrary^® (UPL; Roche Diagnostics) was performed using the web-based software tool ProbeFinder (Roche Diagnostics) (Table 1). Designed primers were purchased from TibMolBiol (Berlin, Germany) and real-time RT-PCR was performed using the LightCycler^® 480 instrument (Roche Diagnostics). PCR assays were prepared using LightCycler^® Probes Master kit (Roche) and the appropriate UPL probe with optimal primers. As housekeeping genes, aminolevulinate synthase 1 (Alas1) and hypoxanthine-guanine phosphoribosyl transferase (Hprt) were used to normalize the gene expression results. Relative quantification was performed by means of the LightCycler^® 480 software v.1.5 (Roche).

Fueldner C., Riemschneider S., Haupt J., Jungnickel H., Schulze F., Zoldan K., Esser C., Hauschildt S., Knauer J., Luch A., Kalkhof S, & Lehmann J. (2022). Aryl Hydrocarbon Receptor Activation by Benzo[a]pyrene Prevents Development of Septic Shock and Fatal Outcome in a Mouse Model of Systemic Salmonella enterica Infection. Cells, 11(4), 737.

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Quantitative Real-Time PCR for IL-1β and G6PD

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For each cDNA sample, the gene expression of IL-1β and the reference gene (G6PD) was analyzed, using a Roche LightCycler 480 II Real-Time PCR instrument (Roche Diagnostics GmbH). The PCR reactions were recorded in a final volume of 20 μL, including 5 μL of cDNA, 3 μL of distilled water, 10 μL of LightCycler 480 Probes Master (Roche Diagnostics GmbH), and 2 μL of primer-probe set (Real-Time Ready single assay; Roche Diagnostics GmbH). The cycle conditions of the relative quantitative PCR (qPCR) were preincubation at 95°C for 10 min, followed by 45 amplification cycles of 95°C for 10 s, at 6°C for 30 s and at 72°C for 1 s, followed by cooling at 40°C for 30 s. The qPCR analysis and the calculation of quantification cycle (Cq) values for relative quantification were performed with the LightCycler 480 Software, v. 1.5 (Roche Diagnostics GmbH). Relative quantitative amounts were calculated by dividing the target genes by the expression level of the reference gene. The reference gene was used for the normalization of the target gene expression.

Ozcicek A., Ozcicek F., Cimen F.K., Mammadov R., Cankaya M., Ezmeci T, & Altuner D. (2018). The effect of anakinra to nephrotoxicity with cisplatin induced in rats: Biochemical, gene expression and histopathological evaluation. Advances in clinical and experimental medicine : official organ Wroclaw Medical University, 27(12).

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