Lightcycler 480 probe master kit

Manufactured by Roche

Sourced in United States, Germany

The LightCycler 480 Probe Master kit is a reagent kit designed for real-time PCR analysis. It contains all the necessary components for the amplification and detection of target DNA sequences using probe-based detection methods. The kit is compatible with the LightCycler 480 instrument.

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Lab products found in correlation

Lightcycler relative quantification software, by Roche (4 mentions) Lightcycler 480, by Roche (4 mentions) Universal probe library probe, by Roche (3 mentions) Transcriptor first strand cdna synthesis kit, by Roche (2 mentions) Lightcycler 480 system, by Roche (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Complete lysis m, by Roche (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Iscript cdna kit, by Bio-Rad (1 mentions) Lightcycler 96 pcr system, by Roche (1 mentions) Epoch spectrophotometer, by Agilent Technologies (1 mentions) Allprep dna rna mini kit, by Qiagen (1 mentions) Universal probe library, by Roche (1 mentions) Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions) Dnase 1, by Thermo Fisher Scientific (1 mentions) High pure rna isolation kit, by Roche (1 mentions) Reverse transcription system a3500, by Promega (1 mentions) Platinum taq dna polymerase high fidelity kit, by Thermo Fisher Scientific (1 mentions) M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Isogen, by Nippon Gene (1 mentions)

15 protocols using lightcycler 480 probe master kit

Western Blotting and qRT-PCR for INS-1E Cells

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After lysing INS-1E cells with a commercial kit (cOm-plete™ Lysis-M, Roche, Merck) the cellular debris was removed after 10 min. 14000 g centrifugation at room temperature. For protein concentration of the extracts, Bradford protein assay was used. Samples (30-50µg) were mixed with a 1×SDS sample buffer (50mM Tris pH6.8, 2% SDS, 10% glycerol, 50mM DTT, and 0.01% bromophenol blue) and were analyzed by using SDS-PAGE on 10% polyacrylamide gels. Then proteins were transferred onto a PVDF membrane (Millipore). After 12 h of incubation with primary antibodies, excess primary antibodies were washed, then, proteins were incubated for an hour at room temperature with secondary antibodies. Finally, membranes were developed via a chemiluminescent detection kit (WBLUF0100, Luminata Millipore).
RNeasy Mini Kit 74104). The quality and quantity of the isolated RNAs were evaluated by Epoch 2.0. and all-inone reader software BioTek Gen 5. Next, mRNAs were converted into cDNAs (Roche Transcriptor First Strand cDNA Synthesis Kit 05081963001). Quantitative Real-Time PCR (qRT-PCR) was carried out using the LightCycler 480 master probe kit (Roche LightCycler 480 master probe kit 04707494001). The real-time ready probes and accession numbers are shown in Table 1. Actb and G6pd were the reference genes that served as internal control. All samples were assayed thrice.

Verdi H., Baysan Cebi H.P., Yilmaz Yalcin Y., Ozkan T., Akcil Ok M., Sunguroglu A, & Atac F.B. (2023). The Role of Parkin in Rat Pancreatic Beta Cells Fate. Cellular and molecular biology (Noisy-le-Grand, France), 69(8).

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Real-time RT-PCR for CRC mRNA Expression

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The LightCycler 480 Probe Master kit (Roche Applied Science, Mannheim, Germany) was used to conduct real-time reverse-transcription (RT) polymerase chain reaction (PCR) to measure the mRNA expression levels. Real-time PCR with specific primers and probe was achieved and detected using the LightCycler 480 Probe Master kit (Roche Applied Science, Indianapolis, IN, USA; Cat. No. 04707494001). The reference gene was glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
The mRNA expression values normalized with GAPDH were calculated using LightCycler Relative Quantification software (Version 2.0, Roche Applied Science). If the mRNA expression level relative to GAPDH in CRC tumor tissue was 1.5-fold higher than that in paired normal colorectal tissue, the mRNA expression was considered high. If the mRNA expression level relative to the control group was 0.5-fold lower, the mRNA expression was considered low. Table S3 and Figure S3A present the primers.

Wang Y.H., Chang S.C., Ansar M., Hung C.S, & Lin R.K. (2021). Eps15 Homology Domain-Containing Protein 3 Hypermethylation as a Prognostic and Predictive Marker for Colorectal Cancer. Biomedicines, 9(5), 453.

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Evaluating SMAD3 mRNA Expression in Colorectal Cancer

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LightCycler 480 (Roche Applied Science, Mannheim, Germany) was the main machine used to gauge mRNA expression and real-time RT-PCR of SMAD3. According to the manufacturer’s guideline, the LightCycler 480 Probe Master Kit (Roche Applied Science, Indianapolis, IN, USA; Cat. no. 04707494001) with specific primers and probe were used to perform real-time PCR. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was the standard for comparison. The PCR conditions were as follows: 95 °C for 10 min and annealing temperature 60 °C for 10 s for a total of 45 cycles. In accordance with the instructions from the manufacturers, GAPDH was used as a reference gene. The normalized gene expression values obtained using LightCycler Relative Quantification software (version 1.5, Roche Applied Science) were then compared with those of the control group. The SMAD3 mRNA expression level was considered high if the mRNA expression level of SMAD3 was twice that of GAPDH in colorectal tumor tissue compared to normal colorectal tissue. Table 3 lists the primers.

Ansar M., Wang C.J., Wang Y.H., Shen T.H., Hung C.S., Chang S.C, & Lin R.K. (2020). SMAD3 Hypomethylation as a Biomarker for Early Prediction of Colorectal Cancer. International Journal of Molecular Sciences, 21(19), 7395.

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Quantification of TMEM240 mRNA Expression

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To measure TMEM240 mRNA expression, real-time RT-PCR was performed with a LightCycler 480 (Roche Applied Science, Mannheim, Germany). Real-time PCR was performed using the LightCycler 480 Probe Master Kit (Roche Applied Science, Indianapolis, IN, USA, cat. no. 04707494001) with specific primers and a corresponding universal library probe (Roche Applied Science) according to the manufacturer’s instructions. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as a reference gene. PCR conditions were as follows: preincubation, 95 °C for 10 min; and amplification, 95 °C for 10 min and 60 °C for 10 min, for a total of 45 cycles. Normalized gene expression values obtained using the LightCycler Relative Quantification software (vers. 1.5, Roche Applied Science) were then compared to those of the control group. TMEM240 mRNA expression was considered low if the mRNA expression level of TMEM240 relative to GAPDH was 0.5-fold lower in colorectal tumor tissues than in paired normal colorectal tissues. The primers and probes used in the RT-PCR are listed in Supplementary Table S1.

Chang S.C., Liew P.L., Ansar M., Lin S.Y., Wang S.C., Hung C.S., Chen J.Y., Jain S, & Lin R.K. (2020). Hypermethylation and decreased expression of TMEM240 are potential early-onset biomarkers for colorectal cancer detection, poor prognosis, and early recurrence prediction. Clinical Epigenetics, 12, 67.

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Quantitative RT-qPCR for Mouse Gene Expression

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RT-qPCR assay was performed using primers for each target gene, which were designed with ProbeFinder version 2.5 software for Mouse Universal ProbeLibrary (Integrated DNA Technologies [IDT: www.idtdna.com]). For each target mRNA gene, 1 μg of total RNA from cardiac tissue was reverse transcribed with the Transcripts First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) and Light Cycler 480 Probe Master Kit (Roche Applied Science, Mannheim, Germany). Cycling conditions were 45 cycles of 95 °C for 10 s, 1 cycle of 60 °C for 30 s and 1 cycle of 72 °C for 1 s. Quantification of relative mRNA levels was calculated from Ct values and normalized to Rplp0 in each sample. The relative expression ratio was calculated by the 2^−ΔΔCt method.

Ballinas-Verdugo M.A., Jiménez-Ortega R.F., Martínez-Martínez E., Rivas N., Contreras-López E.A., Carbó R., Sánchez F., Bojalil R., Márquez-Velasco R., Sánchez-Muñoz F, & Alejandre-Aguilar R. (2021). Circulating miR-146a as a possible candidate biomarker in the indeterminate phase of Chagas disease. Biological Research, 54, 21.

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Evaluating Dendritic Cell Transcripts

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RNA was isolated using AllPrep DNA/RNA Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. Quality and concentration of samples was evaluated using Epoch Spectrophotometer (Bio-Tek, USA). Total RNA was reverse transcribed into cDNA with iScript cDNA Kit (Bio-Rad, USA). For dendritic cells, the evaluation of 14 activity—associated transcripts was conducted with Locked Nucleic Acid probes from the Universal Probe Library—UPL (Roche GmbH Diagnostics, Germany). Real-Time PCR was performed using the LightCycler 480 Probe Master Kit (Roche Diagnostics GmbH, Mannheim, Germany) and Light Cycler^® 96 PCR System (Roche). Reactions were prepared in a total volume of 10 μl. The cycling conditions for UPL reactions were as follows: one cycle of 95°C for 10 min (initial denaturation), 45 cycles of 95°C for 10 s (denaturation) and 60°C for 30 s (annealing). Relative quantities of target genes were determined for unknown samples by the comparative threshold cycle (delta CT) method and normalized to HPRT1 quantities. Gene specific primers and probes sets (Roche Assay ID) used in the experiment are listed in Supplementary Table 1.

Wardowska A., Komorniczak M., Bułło-Piontecka B., Dȩbska-Ślizień M.A, & Pikuła M. (2019). Transcriptomic and Epigenetic Alterations in Dendritic Cells Correspond With Chronic Kidney Disease in Lupus Nephritis. Frontiers in Immunology, 10, 2026.

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Quantifying BEND5 mRNA Expression in Colorectal Cancer

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To measure BEND5 mRNA expression, real-time reverse transcription PCR (RT-PCR) was performed with the LightCycler 480 (Roche Applied Science, Mannheim, Germany). Real-time PCR was performed using the LightCycler 480 Probe Master Kit (Roche Applied Science, Indianapolis, Indiana, USA, Cat. No. 04707494001) with the specific primers and the corresponding Universal Probe Library probe (Roche Applied Science, USA), according to the manufacturer’s instructions. The glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was used as a reference gene. The normalized gene expression values obtained using LightCycler Relative Quantification software (Version 2.0, Roche Applied Science) were then compared with those of the control group. BEND5 mRNA expression was considered low if the mRNA expression level of BEND5 relative to GAPDH was 0.5-fold lower in the colorectal tumor tissue than in the paired normal colorectal tissue. Supplementary Table 5 lists the primers.

Lin R.K., Hung W.Y., Huang Y.F., Chang Y.J., Lin C.H., Chen W.Y., Chiu S.F., Chang S.C, & Tsai S.F. (2017). Hypermethylation of BEND5 contributes to cell proliferation and is a prognostic marker of colorectal cancer. Oncotarget, 8(69), 113431-113443.

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Quantifying gene expression via RT-PCR

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The mRNA expression levels were determined through real-time RT–PCR using a LightCycler 480 (Roche Applied Science, Mannheim, Germany). Real-time RT–PCR was performed using the LightCycler 480 Probe Master kit (Roche Applied Science) and specific CCND2, GAPDH, and other multiple TSG primers and the corresponding Universal Probe Library probe (Roche Applied Science). GAPDH was used as a reference gene. Normalized gene expression values, which were calibrated to the control group, were analyzed while using LightCycler Relative Quantification software (ver. 2.0, Roche Applied Science). The primers are described in Table S6.

Hung C.S., Wang S.C., Yen Y.T., Lee T.H., Wen W.C, & Lin R.K. (2018). Hypermethylation of CCND2 in Lung and Breast Cancer Is a Potential Biomarker and Drug Target. International Journal of Molecular Sciences, 19(10), 3096.

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Stem Cell Marker Evaluation by Real-Time PCR

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Total RNA (1 µg) was reverse transcribed into cDNA with Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany). Evaluation of 27 positive and negative stem cell markers transcripts was conducted with Locked Nucleic Acid probes from the Universal Probe Library – UPL (Roche GmbH Diagnostics, Mannheim, Germany). Real-Time PCR was performed using the LightCycler 480 Probe Master Kit (Roche Diagnostics GmbH, Mannheim, Germany) using Light Cycler 480 v1.5.1 instrument. Reactions were prepared in a total volume of 10 µl. The advanced relative quantification E-method was applied for data analysis, and geometric mean of HPRT1, RPLP0 and RPL13A house-keeping genes expression was used as reference (based on stable expression in ASCs from pentaplicate RNA-seq experiments). Gene specific primers and UPLs used in the experiment are listed in Supplementary Table S2.

Mieczkowska A., Schumacher A., Filipowicz N., Wardowska A., Zieliński M., Madanecki P., Nowicka E., Langa P., Deptuła M., Zieliński J., Kondej K., Renkielska A., Buckley P.G., Crossman D.K., Crowley M.R., Czupryn A., Mucha P., Sachadyn P., Janus Ł., Skowron P., Rodziewicz-Motowidło S., Cichorek M., Pikuła M, & Piotrowski A. (2018). Immunophenotyping and transcriptional profiling of in vitro cultured human adipose tissue derived stem cells. Scientific Reports, 8, 11339.

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Quantification of Runx2 Expression in MC3T3-E1 Cells

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Runx2 gene expression in MC3T3-E1 cells was examined by qPCR. β-actin was used as an internal control gene. The qPCR was performed with a Roche LightCycler 480 system using a LightCycler 480 Probe Master kit (Roche Diagnostics, Basel, Switzerland). Reactions were performed in a final total volume of 20 µl containing 1× LightCycler 480 Probe Master mix, 0.1 µM of each forward and reverse primer, 3.8 µl distilled H₂O and 5 µl of the cDNA template. Specific primers for mouse Runx2 and β-actin were designed using the Universal ProbeLibrary (Roche Diagnostics). The primers used in the current study were as follows: For Runx2, forward, 5′-cgtgtcagcaaagcttctttt-3′ and reverse, 5′-ggctcacgtcgctcatct-3′; and for β-actin, forward, 5′-tgacaggatgcagaaggaga-3′ and reverse, 5′-cgctcaggaggagcaatg-3′. The amplification conditions were as follows: Denaturation at 95°C for 5 min, 45 cycles of amplification (95°C for 10 sec, 60°C for 30 sec and 72°C for 1 sec), and cooling at 50°C for 10 sec. Relative quantification of target gene expression was determined using the E-Method (Efficiency Method) from the LightCycler 480 software (10 (link)).

Wakabayashi Y., Yoshino Y., Seo K., Koga I., Kitazawa T, & Ota Y. (2018). Inhibition of osteoblast differentiation by ritonavir. Biomedical Reports, 9(6), 491-496.

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