Lightcycler 480

Manufactured by Bio-Rad

Sourced in United States

The LightCycler 480 is a real-time PCR instrument designed for quantitative and qualitative nucleic acid analysis. It utilizes a 96-well or 384-well plate format and can accommodate a variety of fluorescent detection chemistries. The system provides thermal cycling capabilities and integrated data analysis software.

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27 protocols using lightcycler 480

Quantifying S1P Pathway Genes by qPCR

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Total RNA was extracted from sorted cell populations using Trizol (Invitrogen) according to the manufacturer’s instructions. Before reverse transcription, RNA was treated with DNase I (Invitrogen). The RNA was converted to cDNA with Invitrogen’s Superscript III First Strand Synthesis System according to the manufacturer’s instructions, using a mix of oligo dT and random hexamers as primers. Quantitative PCR (qPCR) was performed on a Roche Light-Cycler 480 using iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions. Primer pairs used were: Hprt F 5′-AGGTTGCAAGCTTGCTGGT-3′, Hprt R 5′-TGAAGTACTCATTATAGTCAAGGGCA-3′; S1pr5 F 5′-GCCTGGTGCCTACTGCTACAG-3′, S1pr5 R 5′-CCTCCGTCGCTGGCTATTTCC-3′; Spns2 F 5′-AGAAGCCGCATCCTCAGTTAGC-3′, Spns2 R 5′-CAGGCCAGAATCTCCCCAAATC-3′; S1pr1 F 5′-GTGTAGACCCAGAGTCCTGCG-3′, S1pr1 R 5′-AGCTTTTCCTTGGCTGGAGAG-3′; Sphk1 F 5’-CTGGGCTGCGGCTCTATTCTGT-3’; Sphk1 R 5’-AAGGTGCCCACTGTGAAACGAA-3’; Sphk2 F 5’-GTTGTGATCTTGGAGGCTGGT-3’; Sphk2 R 5’-TAGGAACCAAACTCGCCGTG-3’.
To control for DNA contamination, a reaction without reverse transcriptase was performed in parallel for each sample/primer pair. To control for nonspecific amplification, the size of the reaction products was analyzed by agarose gel electrophoresis. Primer pairs were tested for linear amplification over two orders of magnitude.

Baeyens A.A., Bracero S., Chaluvadi V.S., Khodadadi-Jamayran A., Cammer M, & Schwab S.R. (2021). Monocyte-derived S1P in the lymph node regulates immune responses. Nature, 592(7853), 290-295.

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Quantifying Nascent RNA Transcription

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RNA was isolated using the Trizol reagent (Thermo Fisher), and subsequently treated with the DNA-free kit (Thermo Fisher). cDNA was synthesized with the SuperScript III Kit (Thermo Fisher). Nascent RNA extraction was performed using the Click-iT Nascent RNA capture kit (Thermo Fisher) following the manufacture’s protocol. Quantitative PCR was performed on Roche LightCycler 480 real-time PCR machine using the iQ SYBR Green Mastermix (BioRad). Gene expression was analyzed relative to β-actin using the ΔΔCt method. Primer sequences are available upon request.

Liu X., Gogate A.A., Tastemel M., Malladi V.S., Yao H., Nguyen K., Huang L.J, & Bai X. (2017). Dynamic change of transcription pausing through modulating NELF protein stability regulates granulocytic differentiation. Blood Advances, 1(18), 1358-1367.

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Quantification of Gene Expression

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RNA was isolated using Gene MATRIX Universal RNA/miRNA Purification Kit (EURx) and transcribed to cDNA with Transcriptor Universal cDNA Master (Roche). Primer sequences are given in Table S2. Transcript abundance was measured using iTaq Universal SYBR Green Supermix (Bio-Rad) and LightCycler 480. 18S RNA was used as a housekeeping control gene. Relative transcript abundance was assessed using the 2^-ΔΔCT method as previously described [30 (link)].

Białopiotrowicz-Data E., Noyszewska-Kania M., Jabłońska E., Sewastianik T., Komar D., Dębek S., Garbicz F., Wojtas M., Szydłowski M., Polak A., Górniak P, & Juszczyński P. (2023). SIRT1 and HSP90α feed-forward circuit safeguards chromosome segregation integrity in diffuse large B cell lymphomas. Cell Death & Disease, 14(10), 667.

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Quantitative Gene and miRNA Analysis

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RNA and microRNA were isolated using Gene MATRIX Universal RNA/miRNA Purification Kit (EURx, Gdansk, Poland) and transcribed to cDNA with Transcriptor Universal cDNA Master (Roche) and TaqMan MicroRNA Reverse Transcription (Applied Biosystems), respectively. Primer sequences are given in Table S3. Transcript abundance was measured using Itaq Universal SYBR Green Supermix (Bio-Rad) and LightCycler 480. GAPDH and U6 snRNA were used as housekeeping control genes for mRNA and miR-494, respectively. Relative transcript abundance was assessed using the 2^−ΔΔCT method as previously described [46 (link)].

Białopiotrowicz E., Noyszewska-Kania M., Kachamakova-Trojanowska N., Łoboda A., Cybulska M., Grochowska A., Kopczyński M., Mikula M., Prochorec-Sobieszek M., Firczuk M., Graczyk-Jarzynka A., Zagożdżon R., Ząbek A., Młynarz P., Dulak J., Górniak P., Szydłowski M., Pyziak K., Martyka J., Sroka-Porada A., Jabłońska E., Polak A., Kowalczyk P., Szumera-Ciećkiewicz A., Chapuy B., Rzymski T., Brzózka K, & Juszczyński P. (2020). Serine Biosynthesis Pathway Supports MYC–miR-494–EZH2 Feed-Forward Circuit Necessary to Maintain Metabolic and Epigenetic Reprogramming of Burkitt Lymphoma Cells. Cancers, 12(3), 580.

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Macrophage and Endothelial Cell Profiling

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Alveolar and interstitial macrophages as well as endothelial cells (CD31, clone MEC13.3) were sorted into Trizol (Life Technologies, Grand Island, NY) using a Beckman Coulter MoFlow. RNA was isolated and converted into cDNA. cDNA (30 ng) was assessed for DAP12, CXCL1, CXCL2, IL-6, TNFα, and β2 microglobulin (Life Technologies) on a Roche LightCycler® 480 using iTaq Universal Probes Supermix (BioRad, Hercules, CA).

Spahn J.H., Li W., Bribriesco A.C., Liu J., Shen H., Ibricevic A., Pan J., Zinselmeyer B.H., Brody S.L., Goldstein D.R., Krupnick A.S., Gelman A.E., Miller M.J, & Kreisel D. (2015). DAP12 expression in lung macrophages mediates ischemia reperfusion injury by promoting neutrophil extravasation. Journal of immunology (Baltimore, Md. : 1950), 194(8), 4039-4048.

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Quantitative RT-PCR Analysis of SGK1 Expression

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AC-CRISPR cells were lysed in TRIzol Reagent (Life Technologies), rinsed in chloroform, and aqueous layer was applied to Qiagen RNeasy kit (Qiagen; Santa Clarita, CA) columns followed by RNA purification according to manufacturer's instructions. For cDNA synthesis, the Superscript VILO cDNA synthesis kit (Life Technologies) was used according to the manufacturer's instructions, and quantitative RT-PCR (q-RT-PCR) was carried out in triplicate on a Roche (Indianapolis, IN) Lightcycler 480 (software version 1.5.0 SP3) using iQ⁻SYBR Green Supermix (Bio-Rad; Hercules, CA). For human SGK1, two independent primer sets were used including primer set #1 (254bp amplicon): hSGK1 qPCR-1F, 5′-ATGACGGTGAAAACTGAGGCT-3′; hSGK1 qPCR-1R, 5′-GTTCTCCTTGCAGAGTCCGAAG-3′, which were validated by TRC/Broad Institute (primer sequences from PrimerBank, http://pga.mgh.harvard.edu/primerbank/index.html) and primer set #2 (132bp amplicon): hSGK1 qPCR-2F, 5′-GCTGAAATAGCCAGTGCCTTGG-3′; hSGK1 qPCR-2R, 5′-GTTCTCCTTGCAGAGTCCGAAG-3′. Human 18S served as a control using primers: h18S-F, 5′-ACCCGTTGAACCCCATTCGTGA-3′; h18S-R, 5′-GCCTCACTAAACCATCCAATCGG-3′. Melt curves showed single peak specificity for each q-RT-PCR primer set.

Beauchamp R.L., James M.F., DeSouza P.A., Wagh V., Zhao W.N., Jordan J.T., Stemmer-Rachamimov A., Plotkin S.R., Gusella J.F., Haggarty S.J, & Ramesh V. (2015). A high-throughput kinome screen reveals serum/glucocorticoid-regulated kinase 1 as a therapeutic target for NF2-deficient meningiomas. Oncotarget, 6(19), 16981-16997.

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Osteogenic Differentiation of OP-BMSCs

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After osteogenic induction culture of OP-BMSCs (1 × 10⁵ cells/well) in 24-well plates for 14 and 21 days, the expression of runt-related transcription factor-2 (Runx-2), osteopontin (OPN), and receptor activator of NF-κB ligand (RANKL) were investigated using RT-qPCR. Total RNA was collected using TRIzol reagent. The purity of RNA was evaluated by a NanoDrop 2,000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Only an A₂₆₀/A₂₈₀ value around 2.0 was accepted for further analysis. Synthesis of cDNA was performed in a 20-µl reaction volume via a Perfect Real-Time RE reagent kit. Then, the qPCR amplification and detection were analyzed using 2 × Fast SYBR Green Master Mix on a LightCycler 480 (Bio-Rad CFX Manager 3.1, Hercules, CA, USA). Based on the gene expression ratio among different groups, the relative expression level of mRNA was normalized to GAPDH and calculated using the 2^−ΔΔct method. Primer sequences are listed in Supplementary Table S1.

Wang X., Li Z., Wang Z., Liu H., Cui Y., Liu Y., Ren M., Zhan H., Li Z., Wu M, & Wang J. (2021). Incorporation of Bone Morphogenetic Protein-2 and Osteoprotegerin in 3D-Printed Ti6Al4V Scaffolds Enhances Osseointegration Under Osteoporotic Conditions. Frontiers in Bioengineering and Biotechnology, 9, 754205.

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Quantifying S1P Pathway Genes by qPCR

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Baeyens A.A., Bracero S., Chaluvadi V.S., Khodadadi-Jamayran A., Cammer M, & Schwab S.R. (2021). Monocyte-derived S1P in the lymph node regulates immune responses. Nature, 592(7853), 290-295.

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Quantitative gene expression analysis in birds

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RNA extraction was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA samples were extracted from 5 birds tissues and used as a template for reverse transcription. cDNA was obtained by reverse transcriptase SuperScript® III First-Strand Synthesis System using 2 μg total RNA. The relative abundance of mRNA from genes was assessed by real time reverse-transcription (RT)-PCR using a Lightcycler 480 (Bio-Rad) and a Lightcycler 480 SYBR Green I master (Bio-Rad). Primer pairs specific for the amplification of AvBD, cathelicidin and NK-lysin genes are shown in Additional file 1: Table S1. PCR products were subjected to melt curve analysis and sequenced to confirm amplification of the correct gene. Data were analyzed by ddCt method. The mean threshold cycle value (Ct) of each sample was normalized to the internal control, GAPDH, and the expression profiles were obtained by comparing normalized Ct value with the calibrator sample, in which the gene exhibited the lowest expression level. Each analysis was performed in triplicate. Quantification of each sample was calculated with the cycle threshold values and standard curve information using the Lightcycler 480 version 1.5.0 software.

Lee M.O., Jang H.J., Rengaraj D., Yang S.Y., Han J.Y., Lamont S.J, & Womack J.E. (2016). Tissue expression and antibacterial activity of host defense peptides in chicken. BMC Veterinary Research, 12, 231.

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Hamster Splenocyte RNA Extraction

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Splenocytes were collected from hamsters, macerated, lysed in Trizol for RNA extraction. Total RNA was extracted from the ear, lymph node (superficial parotid lymph node), spleen and liver of infected hamsters using TRIzol reagent (Invitrogen). First-strand cDNA synthesis was performed with ≈1–2μg of RNA using a Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Amplification conditions consisted of an initial pre-incubation at 95°C for 10 min, followed by amplification of the target DNA for 40 cycles of 95°C for 15 s and 60°C for 1 min with the LightCycler 480 (Bio-Rad). The efficiency of each reaction was determined. The expression levels of genes of interest were normalized to β-Actin levels. The results are expressed in fold change of 2^-ΔCt over control.

Fiuza J.A., Dey R., Davenport D., Abdeladhim M., Meneses C., Oliveira F., Kamhawi S., Valenzuela J.G., Gannavaram S, & Nakhasi H.L. (2016). Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters. PLoS Neglected Tropical Diseases, 10(1), e0004322.

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