Lightcycler 480 rt pcr system

Manufactured by Roche

Sourced in Switzerland, Germany, China, United States

The LightCycler 480 RT-PCR system is a real-time PCR instrument designed for quantitative and qualitative nucleic acid analysis. It provides accurate and reliable results for a variety of applications, including gene expression analysis, genotyping, and viral load monitoring. The system features a compact design, intuitive software, and a range of advanced detection chemistries to support diverse research needs.

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42 protocols using lightcycler 480 rt pcr system

Quantitative Gene Expression Analysis in Cucurbit

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The seed coat samples from different developmental stages (18 and 26 DAP) and other tissue samples, including roots, stems, leaves, and male flowers were collected from both parental lines. RNA was isolated using the plant total RNA purification kit (TIANGEN, China) according to the manufacturer’s instructions and then the first-strand cDNA was synthesized using a cDNA synthesis kit (Takara, Japan).
The gene-specific primers of the candidate genes and reference gene Actin (Kong et al., 2015 (link)) for quantitative real-time PCR (qRT-PCR) were designed based on the Cucurbit Genomic Database (http://cucurbitgenomics.org), using the software Primer Premier 5. The expression levels of the candidate genes were performed using a LightCycler480 RT-PCR system (Roche, Swiss) with a Real Master Mix (SYBR Green) kit (Toyobo, Japan). Amplification was carried out in a 20 µl reaction mixture containing 1 µl cDNA, 1 µl forward and reverse primers (10 µM), 10 µl 2 × SYBR Green real-time PCR mixes, with nuclease-free water added to a total reaction of 20 µl. Three biological and technical replicates were used for qRT-PCR. Average relative expression levels for each sample were calculated. The expression level was analyzed by the 2^−△△Ct method (Livak and Schmittgen, 2001 (link)), and the primer sequences used in this study are listed in Table S3.

Li B., Lu X., Gebremeskel H., Zhao S., He N., Yuan P., Gong C., Mohammed U, & Liu W. (2020). Genetic Mapping and Discovery of the Candidate Gene for Black Seed Coat Color in Watermelon (Citrullus lanatus). Frontiers in Plant Science, 10, 1689.

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Quantitative Real-Time RT-PCR Analysis

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Total RNA was extracted from 100 mg of root tissue using an RNA extraction kit (Tiangen, Shanghai, China) and reverse transcribed using a ReverTra Ace qRT-PCR kit (Toyobo, Tokyo, Japan) according to the manufacturer’s instructions. The qRT-PCR was performed using the LightCycler 480 RT PCR system (Roche, Basel, Switzerland), as described earlier (Wang et al., 2019 (link)). The PCR program was performed using pre-denaturation at 94°C for 3 min, followed by 40 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, and then a final extension at 72°C for 5 min. Relative gene expression was calculated as described previously (Livak and Schmittgen, 2001 (link)). Three biological replicates were analyzed. The primers specific for target genes and the internal control ACTIN gene are presented in Supplemental Table S2.

Shang Y., Wang K., Sun S., Zhou J, & Yu J.Q. (2019). COP9 Signalosome CSN4 and CSN5 Subunits Are Involved in Jasmonate-Dependent Defense Against Root-Knot Nematode in Tomato. Frontiers in Plant Science, 10, 1223.

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Quantitative gene expression analysis

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RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's instructions. RNA was treated with DNAse I (Roche Diagnostics, Meylan, France) to remove any contaminating DNA. A total of 1 µg RNA was reverse-transcribed using SuperScript II (Invitrogen; Thermo Fisher Scientific, Inc.). The target TH, DCX and PHOX2B gene transcripts and the reference β-2-microglobulin (β2M) housekeeping gene transcript were measured using the LightCycler 480 RT-PCR system (Roche Diagnostics), using previously reported primers and probes (24 (link)). The sequences are provided in supplementary methods of the publication (24 (link)). The cycling conditions were as follows: 10 min at 95°C, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Absolute quantification analysis with the LightCycler software was performed to determine transcripts numbers, as described previously (20 (link)). Briefly, standard curves were generated using serial dilutions of plasmid containing a known number of molecules of each transcript and used to calculate the number of transcripts in the samples. The number of the reference β2M gene transcripts was used for the normalization of data. Normalized data were expressed as the copy numbers of target transcripts per 10⁶ copies of β2M transcripts. The PCR reactions were performed in triplicate.

Grèze V., Kanold J., Chambon F., Halle P., Gremeau A.S., Rives N., Rouel N., Pereira B., Tchirkov A, & Brugnon F. (2017). RT-qPCR for PHOX2B mRNA is a highly specific and sensitive method to assess neuroblastoma minimal residual disease in testicular tissue. Oncology Letters, 14(1), 860-866.

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RT-qPCR Analysis of Apoptosis-Related Genes

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Total RNA was extracted using Trizol reagent. Reverse transcription and SYBR green qPCR was performed using a kit from Vazyme lnc, Nanjing China. and carried out by the Roche LightCycler 480 RT-PCR System. β-actin was placed as a loading control, and the delta delta Ct (2^−ΔΔCt) method was used to calculate the fold changes. Primers were used from the literature, as shown in the following [60 (link)]: BCL-w F: AGT TCG AGA CCC GCT TCC R: CCC GTC CCC GTA TAG AGC BCL-xl F: CTG AAT CGG AGA TGG AGA CC R: TGG GAT GTC AGG TCA CTG AA ACTB F: GCC CTG AGG CAC TCT TCC A R: CCA GGG CAG TGA TCT CCT TCT.

Liu Y., Yang H., Liu Q., Pan M., Wang D., Pan S., Zhang W., Wei J., Zhao X, & Ji J. (2022). Selenocystine-Derived Label-Free Fluorescent Schiff Base Nanocomplex for siRNA Delivery Synergistically Kills Cancer Cells. Molecules, 27(4), 1302.

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Validating RNA-seq Data Using qRT-PCR

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To validate the RNA-seq results, water-stressed tomato leaf samples from each sampling time point were subjected to qRT-PCR analysis. Total RNA was provided by Gene Denovo Biological Technology Co., Ltd. (Guangzhou, China). The cDNA was reverse-transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, China), following the protocol of the manufacturer. Gene-specific qRT-PCR primers were designed using Primer-BLASÉ in National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) (accessed on 10 January 2019) for 13 selected genes. qRT-PCR was performed using a LightCycler-480 RT-PCR system (Roche, Basel, Switzerland). Each reaction mixture contained 5 µL 2 × TB Green Master Mix Reagent (Takara, China), 1 µL cDNA sample, and 100 nM gene-specific primer in a final volume of 10 µL. PCR conditions were as follows: 95 °C for 30 s, followed by 40 cycles of heating at 95 °C for 5 s and annealing at 60 °C for 30 s. A template-free control for each primer pair was set for each cycle. All PCR reactions were normalized using the Ct value corresponding to the tomato UBI gene. Measurements of three biological and three technical replicates were used.

Chen M., Zhu X., Liu X., Wu C., Yu C., Hu G., Chen L., Chen R., Bouzayen M., Zouine M, & Hao Y. (2021). Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit. International Journal of Molecular Sciences, 22(7), 3347.

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Quantifying Gene Expression in Cochlear Cells

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Total RNA was extracted from OC-1 cells or whole cochleae with ExTrizol Reagent (Protein Biotechnology, PR90) and reverse transcribed to cDNA by using cDNA Synthesis Kits (Thermo Fisher Scientific, K1622) according to the manufacturers’ protocols. The qRT-PCR was performed on a LightCycler 480 RT-PCR system (Roche Diagnostics Ltd, Switzerland) with the LightCycler 480 SYBR Green I Master (Roche Diagnostics, 04887352001). Validated primers were designed for each targeted mRNA. qPCR conditions consisted of an initial denaturing step of 5 min at 95°C followed by 45 cycles of 10 s denaturation at 95°C, 20 s annealing at 60°C, and 20 s extension at 72°C. The mRNA expression was normalized to the mRNA expression of GAPDH. The results were calculated using the comparative cycle threshold (ΔΔCt) method.

He Z.H., Li M., Fang Q.J., Liao F.L., Zou S.Y., Wu X., Sun H.Y., Zhao X.Y., Hu Y.J., Xu X.X., Chen S., Sun Y., Chai R.J, & Kong W.J. (2021). FOXG1 promotes aging inner ear hair cell survival through activation of the autophagy pathway. Autophagy, 17(12), 4341-4362.

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Quantitative Gene Expression Analysis

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Total mRNA of samples was extracted with an RNA Simple Total RNA kit (Tiangen, Beijing, China). Reverse transcription was performed using random 6-mer and oligo dT primers with a PrimeScript RT reagent kit (Takara, Dalian, China). The qRT-PCR was carried out with SYBR Premix Ex Taq II (Takara) following the LightCycler 480 RT-PCR System protocol (Roche, Basel, Switzerland). The qRT-PCR analysis was repeated three times for each sample. The qRT-PCR primers are listed in Supplementary Table S3.

Gu P., Su T., Wang Q., Liang Q, & Qi Q. (2016). Tunable switch mediated shikimate biosynthesis in an engineered non-auxotrophic Escherichia coli. Scientific Reports, 6, 29745.

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Quantifying Gene Expression in Lipid Metabolism

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Total RNA was extracted using an RNeasy Kit (Qiagen) according to the manufacturer’s instructions. RNA was transcribed into complementary DNA (cDNA) using SuperScript II reverse transcriptase (Invitrogen). Real-time PCR (RT-PCR) was performed in a LightCycler 480 RT-PCR System (Roche Diagnostics GmbH). Primers for human PCYT2 (F-5′-CTCACCACAGACCTCATCGT-3′, R-5′-TGCCAGGTTAGAAGTCACCA-3′), SREBP-1c (F-5′-GGAGGGGTAGGGCCAACGGCCT-3′, R-5′-CATGTCTTCGAAAGTGCAATCC-3′), and FASn (F-5′-CGCGTGGCCGGCTACTCCTAC-3’, R-5′-CGGCTGCCACACGCTCCTCT-3′) were designed (Invitrogen). Standardized primers for human DGAT1 (HS00201385_M1), DGAT2 (HS01045913_M1), PLA₂G6 (Hs00895669_m1) and RPLP0 (HS99999902_M1) were used (Custom TaqMan Gene Expression Assays; Applied Biosystems).
Each sample was run in triplicate, and the mean value was used to calculate the mRNA expression using the comparative (2−ΔCt) method according to the manufacturer’s instructions.

Launay N., Ruiz M., Planas-Serra L., Verdura E., Rodríguez-Palmero A., Schlüter A., Goicoechea L., Guilera C., Casas J., Campelo F., Jouanguy E., Casanova J.L., Boespflug-Tanguy O., Vazquez Cancela M., Gutiérrez-Solana L.G., Casasnovas C., Area-Gomez E, & Pujol A. (2023). RINT1 deficiency disrupts lipid metabolism and underlies a complex hereditary spastic paraplegia. The Journal of Clinical Investigation, 133(14), e162836.

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Quantitative Analysis of mRNA Expression

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Total RNA was extracted from cells and adipose tissues using TRIzol (Invitrogen, Grand Island, NY). cDNA synthesis and quantitative RT-PCR were performed as previously reported with some modifications29 . SYBR Green I Master mix (Roche, Indianapolis, IN) and a Roche LightCycler 480 RT-PCR system were used. The primers used for PCR are described in Supplementary Table S1. The expression levels of the mRNAs were normalized to the expression level of 36B4 and then compared.

Kim J.M., Jang H.J., Choi S.Y., Park S.A., Kim I.S., Yang Y.R., Lee Y.H., Ryu S.H, & Suh P.G. (2014). DJ-1 contributes to adipogenesis and obesity-induced inflammation. Scientific Reports, 4, 4805.

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Quantitative Real-Time PCR Protocol

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The total RNA from collected cells was subjected to RNA extraction using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA. A quantitative real-time PCR (qPCR) test was completed using a Lightcycler 480 RT-PCR system (Roche Diagnostics; Mannheim, Germany) with a SYBR Green I Master RT-PCR kit (Roche Diagnostics). Relative quantitation was performed based on the ^ΔΔCT method. The primers used for the qPCR are shown in Table 1.

Meng Y., Li C., Liang Y., Jiang Y., Zhang H., Ouyang J., Zhang W., Deng R., Tan Q., Yu X, & Luo Z. (2023). Umbilical Cord Mesenchymal-Stem-Cell-Derived Exosomes Exhibit Anti-Oxidant and Antiviral Effects as Cell-Free Therapies. Viruses, 15(10), 2094.

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