Lightcycler technology

Manufactured by Roche

Sourced in Germany, Switzerland

The LightCycler technology is a real-time PCR (Polymerase Chain Reaction) platform developed by Roche. It is designed for the detection and quantification of nucleic acids, enabling rapid and accurate analysis of DNA and RNA samples. The LightCycler system provides a closed-tube format and utilizes fluorescent dyes or probes to monitor the amplification of target sequences in real-time during the PCR process.

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Lab products found in correlation

Verso cdna synthesis kit, by Thermo Fisher Scientific (3 mentions) High pure rna isolation kit, by Roche (3 mentions) Amplicon kit, by Roche (3 mentions) Primer express software, by Thermo Fisher Scientific (3 mentions) Rneasy rna purification kit, by Qiagen (3 mentions) Lightcycler 480 dna sybr green 1 master kit, by Roche (2 mentions) Lightmix apoe c112r r158c kit, by TIB Molbiol (2 mentions) Fast start dna master hybprobe kit, by Roche (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Iscript kit, by Bio-Rad (2 mentions) Quantitect primer assay, by Qiagen (2 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Human gene 1.0 st array, by Thermo Fisher Scientific (1 mentions) Trifast, by Avantor (1 mentions) Rlt buffer, by Qiagen (1 mentions) Rneasy kit, by Qiagen (1 mentions) Taqman assay, by Thermo Fisher Scientific (1 mentions) Innotest phospho tau 181p, by Fujirebio (1 mentions) Qiaamp dna blood mini kit, by Qiagen (1 mentions) Fast start sybr green 1 kit, by Roche (1 mentions)

Close spelling variants of this product

LightCycler (1119 citations) LightCycler Technology (LC-96; (2 citations) Lightcycler-480 PCR technology (2 citations)

41 protocols using lightcycler technology

RNA Isolation and Microarray Analysis

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Isolation of total cellular RNA from cultured cells and tissues and reverse transcription were performed as described [10 (link),65 (link)]. 300 ng of RNA were hybridized to Affymetrix Human Gene ST 1.0 arrays following the standard Affymetrix protocol (Affymetrix, High Wycombe, UK). Hybridization and scanning were performed at an Affymetrix Service Provider and Core Facility, “KFB—Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany; www.kfb-regensburg.de).
Quantitative real-time-PCR was performed applying LightCycler technology (Roche, Mannheim, Germany) and the following pairs of primers: human PAPPA (forward: 5'-AGC CAG CAG CAT CCC AGG TGT-3'; reverse: 5'-CGC CCG GAG CCA AAA AGT GGT)-3' and human collagen type I (forward: 5'- CGG CTC CTG CTC CTC TT -3'; reverse: 5'-GGG GCA GTT CTT GGT CTC -3'). Amplification of cDNA derived from 18s rRNA (forward: 5'-TCT GTG ATG CCC TTA GAT GTC C-3'; reverse: 5'-CCA TCC AAT CGG TAG TAG CG-3') was used for normalization.

Engelmann J.C., Amann T., Ott-Rötzer B., Nützel M., Reinders Y., Reinders J., Thasler W.E., Kristl T., Teufel A., Huber C.G., Oefner P.J., Spang R, & Hellerbrand C. (2015). Causal Modeling of Cancer-Stromal Communication Identifies PAPPA as a Novel Stroma-Secreted Factor Activating NFκB Signaling in Hepatocellular Carcinoma. PLoS Computational Biology, 11(5), e1004293.

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RNA Extraction and qPCR Analysis

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RNA was purified from homogenized tissues (Precellys, VWR International, Radnor, PA) with TriFast (VWR International) according to the manufacturer´s instructions. Stress stimulated cells were lysed with 300 μL RLT buffer (Qiagen, Hilden, Germany) and RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer’s protocol. RNA was reverse-transcribed to cDNA with the Iscript^TM Kit (Biorad, Hercules, CA). The qPCR was performed using the LightCycler® technology (LC480) and the LightCycler 480 DNA SYBR Green I Master Kit (Roche Applied Science) according to the manufacturer’s protocol. The following primers were used: b2m-f (5´-gatgagtatgcctgccgtgtg-3´) and b2m-r (5´-caatccaaatgcggcatct-3´) for the housekeeping gene Beta-2 microglobulin (B2M); SESN1-f (5´-agcccatagaccttggctta-3´) and SESN1-r (5´-tccacactgtgattgccatt-3´) for SESN1; SESN2-f (5´-tgctgtgctttgtggaagac-3´) and SESN2-r (5´-gctgcctggaacttctcatc-3´) for SESN2.

Mlitz V., Gendronneau G., Berlin I., Buchberger M., Eckhart L, & Tschachler E. (2016). The Expression of the Endogenous mTORC1 Inhibitor Sestrin 2 Is Induced by UVB and Balanced with the Expression Level of Sestrin 1. PLoS ONE, 11(11), e0166832.

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Cerebrospinal Fluid Biomarker Analysis

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Lumbar puncture was performed before noon, and CSF was collected in polypropylene tubes (Thermo Nunc) and centrifuged within 4 h at 2000 g for 10 min at room temperature. The supernatant was transferred to new tubes and frozen at –80°C prior to analysis. All CSF samples were analyzed at the Department of Interdisciplinary Laboratory Medicine and Medical Biochemistry at Akershus University Hospital, and samples from other sites were frozen before sending to this laboratory. CSF Aβ₄₂, total tau, and phosphorylated tau were determined using ELISA (Innotest β-Amyloid (1–42), Innotest h-Tau Ag and Innotest Phospho-Tau (181P), Fujirebio, Ghent, Belgium).
APOE genotyping was performed on EDTA blood samples either at Akershus University Hospital (Gene Technology Division, Department of Interdisciplinary Laboratory Medicine and Medical Biochemistry) according to the laboratory’s routine protocol using real-time PCR combined with a TaqMan assay (Applied Biosystems, Thermo Fisher Scientific, Waltham, USA) or at the University Hospital of Trondheim according to the protocol for the Fast Start DNA Master HybProbe Kit (Roche, Basel, Switzerland) in combination with the LightMix ApoE C112R R158C kit from TiB MolBiol (Berlin, Germany) followed by LightCycler technology (Roche, Basel, Switzerland).
All subjects were referred to an MRI scan, and if available also to FDG-PET and amyloid PET.

Fladby T., Pålhaugen L., Selnes P., Waterloo K., Bråthen G., Hessen E., Almdahl I.S., Arntzen K.A., Auning E., Eliassen C.F., Espenes R., Grambaite R., Grøntvedt G.R., Johansen K.K., Johnsen S.H., Kalheim L.F., Kirsebom B.E., Müller K.I., Nakling A.E., Rongve A., Sando S.B., Siafarikas N., Stav A.L., Tecelao S., Timon S., Bekkelund S.I, & Aarsland D. (2017). Detecting At-Risk Alzheimer’s Disease Cases. Journal of Alzheimer's Disease, 60(1), 97-105.

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APOE Genotyping Protocol for Patient Samples

Cited 1 time

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APOE genotyping was performed on blood samples from all patients and control individuals. Fresh whole blood was drawn into 6 mL EDTA-vacutainers, and DNA isolated using the QIAamp DNA Blood Mini Kit (QIAGEN), together with the spin protocol provided. Random samples of isolated DNA were checked for purity using NanoDrop technology, and all samples were frozen and stored at −80°C. APOE analysis was either performed according to the protocol described elsewhere5 (link) or using the Fast Start DNA Master HybProbe Kit (Roche) in combination with the LightMix ApoE C112R R158C kit from TiB MolBiol (Berlin, Germany) according to the manufacturer's instructions, followed by APOE genotyping with LightCycler technology (Roche).

Berge G., Sando S.B., Rongve A., Aarsland D, & White L.R. (2014). Apolipoprotein E ε2 genotype delays onset of dementia with Lewy bodies in a Norwegian cohort. Journal of Neurology, Neurosurgery, and Psychiatry, 85(11), 1227-1231.

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Quantitative Transcriptional Analysis

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Total RNA was extracted from stomachs or cell cultures with the HighPure RNA Isolation kit (Roche). Reverse transcription was performed using the Verso cDNA synthesis kit (Thermo Scientific) and RT-qPCR was performed using LightCycler technology (Roche Diagnostics). PCR primers (Additional file 11: Table S2) were designed using the LightCycler Probe Design 2.0 software. Each sample was analysed in three independent experiments done in triplicate. Expression levels were determined with the LightCycler analysis software (version 3.5) relative to standard curves. Data were represented as the mean level of gene expression relative to the expression of the reference genes UBIQUITIN or GAPDH. Relative mRNA expression was calculated using the 2^–ΔΔCT method [47 (link)].

McKey J., Martire D., de Santa Barbara P, & Faure S. (2016). LIX1 regulates YAP1 activity and controls the proliferation and differentiation of stomach mesenchymal progenitors. BMC Biology, 14, 34.

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Quantitative PCR Protocol for Gene Expression

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RNA isolation from cells and tissues and subsequent reverse transcription were performed as described in [32 (link)]. Quantitative real-time PCR was performed by applying LightCycler technology (Roche Diagnostics, Mannheim, Germany) while using specific sets of primers, as listed in Table 1. For normalization, the amplification of cDNA derived from β-actin was carried out.

Sommer J., Ehnis H., Seitz T., Schneider J., Wild A.B., Moceri S., Buechler C., Bozec A., Weber G.F., Merkel S., Beckervordersandforth R., Steinkasserer A., Schüle R., Trebicka J., Hartmann A., Bosserhoff A., von Hörsten S., Dietrich P, & Hellerbrand C. (2023). Four-and-a-Half LIM-Domain Protein 2 (FHL2) Induces Neuropeptide Y (NPY) in Macrophages in Visceral Adipose Tissue and Promotes Diet-Induced Obesity. International Journal of Molecular Sciences, 24(19), 14943.

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Quantitative RNA Expression Analysis

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Total RNA from cells was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA was reverse transcribed into cDNA using the Omniscript RT kit (Qiagen). RT‐qPCR was performed using LightCycler technology (Roche, Germany) and the Fast Start SYBR Green I kit. The expression of the target molecule was normalized to GAPDH. The primer sets used (all from Sigma‐Aldrich) are listed in Table S1.

Fischer A., Abdollahi‐Roodsaz S., Böhm C., Niederreiter B., Meyer B., Yau A.C., Lönnblom E., Joosten L.A., Koenders M., Lehmann C.H., Dudziak D., Krönke G., Holmdahl R, & Steiner G. (2018). The involvement of Toll‐like receptor 9 in the pathogenesis of erosive autoimmune arthritis. Journal of Cellular and Molecular Medicine, 22(9), 4399-4409.

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Gene Expression Analysis of Inflammatory Markers

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RNX-plus kit (Cinnacolon, Tehran, Iran) was used to do RNA extraction. RNA suspension was frozen in −20 °C until it was converted to cDNA. Following the extraction of the total RNAs from each sample, RNA quantification were performed using UV spectrophotometer. Each sample OD 260/280 ratio was intended between 1.7 and 2.1, demonstrating no contamination with both protein and DNA [25 (link)]. The isolated RNA was reverse transcribed to cDNA library, using moloney murine leukemia virus (MMLV) reverse transcriptase (RT). Gene expression of PPAR-γ, LDLR, IL-1, IL-8, and TNF-α were evaluated by quantitative RT-PCR in PBMCs, using the LightCycler technology (Roche Diagnostics, Rotkreuz, Switzerland) with SYBR green detection and Amplicon Kit (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used as housekeeping gene. To design primers, Primer Express Software (Applied Biosystems, Foster City, CA, USA) and Beacon designer software (Takaposizt, Tehran, Iran) were used. Relative transcription levels were calculated using the method of Pffafi or 2^−∆∆CT.

Jamilian M., Samimi M., Mirhosseini N., Afshar Ebrahimi F., Aghadavod E., Taghizadeh M, & Asemi Z. (2018). A Randomized Double-Blinded, Placebo-Controlled Trial Investigating the Effect of Fish Oil Supplementation on Gene Expression Related to Insulin Action, Blood Lipids, and Inflammation in Gestational Diabetes Mellitus-Fish Oil Supplementation and Gestational Diabetes. Nutrients, 10(2), 163.

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RNA Extraction and qPCR Analysis of Mouse Cells

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Total RNA was isolated from mouse sorted Venus+ α cells using RNeasy Plus Micro kit (Qiagen). After specific quantification (Qubit RNA High-sensitivity Assay Kit, life technologies) 10ng of total RNA were reverse-transcribed (PrimeScript RT reagent Kit, Takara) and preamplified (cDNA Pre-Amp Master kit, Roche Diagnostics) following the manufacturer’s instructions. Through candidate gene approach, specific mRNA levels were analyzed by real-time qPCR using Light-cycler technology (Roche Diagnostics). The expression of each candidate gene expression was evaluated using specific primers (Supplemented data, Table 1-3).

Dusaulcy R., Handgraaf S., Heddad-Masson M., Visentin F., Vesin C., Reimann F., Gribble F., Philippe J, & Gosmain Y. (2015). Alpha-cell dysfunctions and molecular alterations in male insulinopenic diabetic mice are not completely corrected by insulin. Endocrinology, 157(2), 536-547.

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Comprehensive Iron Biomarker Profiling

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Serum iron, transferrin, glucose, C‐reactive protein (CRP), liver function tests, lipids, haemoglobin (Hgb) and serum ferritin were measured using routine methods. HFE genotyping was performed using LightCycler technology (Roche Diagnostics) with Genes‐4U toolsets (Ratiogen, AG).
Serum hepcidin was measured using the high sensitivity enzyme immunoassay ELISA kit (Hepcidin 25 (bioactive), EIA‐5782, DRG Diagnostics, Marburg, Germany). Thawed samples (one thaw only) were analysed for hepcidin between April and September 2019. Low and high controls provided in the kit were run with each calibration curve, and results were considered invalid if the assay did not fit the acceptable ranges of the low and high controls. The assay range is between 0.153 ng/ml and 81 ng/ml. The limit of detection is 0.304 ng/ml. NTBI was measured using a nitrilotriacetic (NTA) assay [19 (link)]. The lower limit of detection was <0.47 µM. For those samples that received a <0.47 µM value, a value of 0.46 was entered onto the SPSS database. NTBI was measured from October to December 2020.

Ryan E., Mulready K., Wiegerinck E., Russell J., Swinkels D.W, & Stewart S. (2022). NTBI levels in C282Y homozygotes after therapeutic phlebotomy. EJHaem, 3(3), 644-652.

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