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Lightcycler 480 instrument 2 real time pcr system

Manufactured by Roche
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Sourced in Switzerland, Germany

The LightCycler 480 Instrument II is a real-time PCR system designed for quantitative nucleic acid analysis. It utilizes optical detection technology to monitor the amplification of DNA or RNA targets in real-time. The instrument provides precise temperature control and advanced data analysis capabilities to support various real-time PCR applications.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (7 mentions) Sybr green real time pcr master mix kit, by Toyobo (2 mentions) Revertra ace qpcr rt kit, by Toyobo (2 mentions) Nuclease free water, by New England Biolabs (2 mentions) Lightcycler 480 sybr green 1 master mix, by Roche (2 mentions) Cell recovery solution, by Corning (2 mentions) Quick rnatm miniprep, by Zymo Research (2 mentions) Biorad dna engine, by Bio-Rad (1 mentions) Reverse transcription kit, by Takara Bio (1 mentions) Trizol ls reagent, by Thermo Fisher Scientific (1 mentions) Sybr premix ex taq 2 kit, by Takara Bio (1 mentions) Nucleospin rna 2, by Takara Bio (1 mentions) Transcriptor universal cdna master, by Roche (1 mentions) Taqman genotyping assay, by Thermo Fisher Scientific (1 mentions) Sybr green 1 master mix, by Roche (1 mentions) High capacity cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Primer premier v6, by Premier Biosoft (1 mentions) Tb green premix ex taq kit, by Takara Bio (1 mentions) Beacon designer 7, by Premier Biosoft (1 mentions) Revertra ace qpcr rt master mix, by Toyobo (1 mentions)

Close spelling variants of this product

LightCycler 480 II Real-Time PCR System LightCycler 480 Real-Time PCR System LightCycler 480 II real-time PCR LightCycler® 480 II Real-Time System LightCycler 480 Real-Time PCR LightCycler 480 Real-Time System Roche 480 real-time PCR system LightCycler 480 II PCR system Real-Time PCR LightCycler II LightCycler 480 PCR system

16 protocols using lightcycler 480 instrument 2 real time pcr system

1

qPCR Analysis of Skin Marker Genes

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RNA extraction and cDNA synthesis were conducted as previously described [16 (link), 26 (link)]. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed with the LightCycler 480 Instrument II Real-Time PCR System (Roche Diagnostics Ltd., Rotkreuz, Switzerland). The relative expression of each mRNA was normalized to β-actin as an internal control. The primers used in this study were as follows: IVL: forward 5′-TCCTCCAGTCAATACCCATCAG-3′; reverse 5′-CAGCAGTCATGTGCTTTTCCT-3′; CK13: forward 5′-CCCCAGGCATTGACCTGAC-3′; reverse 5′-GTGTTGGTAGACACCTCCTTG-3′; ACTB: forward 5′-TTGTTACAGGAAGTCCCTTGCC-3′; reverse: 5′-ATGCTATCACCTCCCCTGTGTG-3′.
Yoshioka M., Ohashi S., Ida T., Nakai Y., Kikuchi O., Amanuma Y., Matsubara J., Yamada A., Miyamoto S., Natsuizaka M., Nakagawa H., Chiba T., Seno H, & Muto M. (2017). Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells. Journal of Experimental & Clinical Cancer Research : CR, 36, 101.
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2

Quantitative Real-Time PCR Assay Protocol

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Total RNAs were extracted using TRIzol reagent (ThermoFisher). First-strand cDNAs were synthesized using a ReverTra Ace qPCR RT kit (TOYOBO). Conventional PCR was carried out in a BioRad DNA Engine (BioRad). Real time PCR was carried out in a LightCycler 480 Instrument II real-time PCR system (Roche), using a SYBR Green Realtime PCR Master Mix kit (TOYOBO). The relative amounts of transcripts were calculated using the 2−ΔΔCt formula (Schmittgen and Livak, 2008 (link)), normalized to GAPDH or beta-actin transcript as an internal control. PCR primers were listed in Table S1.
Du J., Liao W., Liu W., Deb D.K., He L., Hsu P.J., Nguyen T., Zhang L., Bissonnette M., He C, & Li Y.C. (2020). N6-Adenosine methylation of Socs1 mRNA is required to sustain the negative feedback control of macrophage activation. Developmental cell, 55(6), 737-753.e7.
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3

Quantitative PCR Analysis of Gene Expression

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CP RNA for each animal was collected and transcribed as described for RT-PCR. The cDNA was diluted with nuclease-free water (New England Biolabs). A standard curve of 1:10, 1:100, 1:1000, and 1:10000 dilutions of cDNA was used to determine the linear rage of the qPCR assay using the described primer pairs. All samples were run in triplicate. qPCR was performed with a LightCycler 480 Instrument II real-time PCR system (Roche LifeScience), using LightCycler 480 SYBR Green I Master Mix (Roche LifeScience, 04707516001). qPCR cycle conditions were 95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. Data are displayed as a fold change in expression using the 2–ΔΔCT method, relative to the calibrator reference genes GAPDH and RPS18. Data are shown as fold change relative to the normalized control (untreated normal animals).
Hochstetler A., Smith H., Reed M., Hulme L., Territo P., Bedwell A., Persohn S., Perrotti N., D’Antona L., Musumeci F., Schenone S, & Blazer-Yost B.L. (2023). Inhibition of serum- and glucocorticoid-induced kinase 1 ameliorates hydrocephalus in preclinical models. Fluids and Barriers of the CNS, 20, 61.
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4

Quantifying miR-503 Expression in Cells

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Total RNA was isolated using TRIzol LS reagent according to the manufacturer’s instructions (Invitrogen), and then reverse-transcribed with the Reverse Transcription kit (Takara) to generate first-strand miRNA-cDNA PCR templates. Quantitative real-time PCR was performed using SYBR Premix Ex Taq II kit (Takara, Otsu, Japan) on the LightCycler 480 Instrument II Real-time PCR System (Roche, Basel, Switzerland). All PCR experiments were performed in triplicate. Primers specific for human miR-503 were used, and miR-39 was used as an internal control. Relative miRNA expression was quantified with the –ΔΔCt method, and the fold-change was determined using the formula 2–ΔΔCt.28 (link)
Yue H.Q., Zhou Y.H., Guo Y., Tang C.Y., Wang F, & Zhou H.D. (2020). Serum miR-503 is a Candidate Biomarker for Differentiating Metabolic Healthy Obesity from Metabolic Unhealthy Obesity. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 13, 2667-2676.
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5

RNA Extraction and Gene Expression Analysis

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Total cellular RNA samples were isolated from mouse colon tissues and from HCT116 cells using NucleoSpin RNA II (Takara). The cDNAs were generated from 1-μg total RNA by reverse transcription using Transcriptor Universal cDNA Master (Roche, Branchburg, NJ, USA). The mRNA expression levels of mouse Atg5, interleukin (IL) 1-β, chemokine (C-X-C motif) ligand 1 (CXCL1), p53 upregulated modulator of apoptosis (PUMA), Noxa, Bax, CHOP, BiP, spliced X-box binding protein 1 (sXBP1) and of human Atg5, Ulk1, Atg7, C-X-C chemokine receptor type 4 (CXCR4), SOX9, CD44, CXCL1, IL8, cellular inhibitor of apoptosis protein 1 (cIAP1), PUMA, Noxa, Bax, CHOP, BiP, and spliced XBP1 were determined by quantitative real-time RT-PCR using the LightCycler 480 instrument II real-time PCR System (Roche). GAPDH mRNA was used as an internal control. The primer sequences used are available on request.
Sakitani K., Hirata Y., Hikiba Y., Hayakawa Y., Ihara S., Suzuki H., Suzuki N., Serizawa T., Kinoshita H., Sakamoto K., Nakagawa H., Tateishi K., Maeda S., Ikenoue T., Kawazu S, & Koike K. (2015). Inhibition of autophagy exerts anti-colon cancer effects via apoptosis induced by p53 activation and ER stress. BMC Cancer, 15, 795.
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6

Quantitative Gene Expression Analysis

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Total RNAs were extracted using TRIzol reagent (ThermoFisher). First-strand cDNAs were synthesized using a ReverTra Ace qPCR RT kit (TOYOBO). Real time PCR was carried out in a LightCycler 480 Instrument II real-time PCR system (Roche), using a SYBR Green Realtime PCR Master Mix kit (TOYOBO). The relative amounts of transcripts were calculated using the 2−ΔΔCt formula (Schmittgen and Livak, 2008 (link)), normalized to GAPDH transcript as an internal control. PCR primers were listed in Table S2.
Du J., Sarkar R., Li Y., He L., Kang W., Liao W., Liu W., Nguyen T., Zhang L., Deng Z., Dougherty U., Kupfer S.S., Chen M., Pekow J., Bissonnette M., He C, & Li Y.C. (2022). N6-Adenomethylation of GsdmC is essential for Lgr5+ stem cell survival to maintain normal colonic epithelial morphogenesis. Developmental cell, 57(16), 1976-1994.e8.
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7

Quantitative PCR Analysis of Breast Cancer Spheroids

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The spheroids were treated and harvested with Cell Recovery Solution (Corning Incorporated, Corning, NY, USA) to remove the Matrigel. Total RNA was extracted from MCF7, MCF7TR, Sap-treated MCF7TR spheroids using Quick -RNATM Mini Prep (Zymo Research, USA) according to the manufacturer’s instructions. Each 10 μL reaction consists of Superscript III RT/Platinum Taq mix, 5ul of 2X SYBR green reaction mix, 1 ul of test primer, 300ng RNA and distilled water. Quantitative Real-time PCR was performed on Light Cycler® 480 Instrument II real-time PCR system (Roche Diagnostics, Penzberg, Germany) and Ct values were outputted for quantification. Initial enzyme activation was performed at 95C for 15 min, followed by 70 cycles of denaturation at 95C for 15 s and primer annealing/extension at 60C for 70 s. Melting curve analysis was performed at 95C for 5 s, 65C for 60 s, and 45C for 30 s. Primers used are listed in Supplementary Table S4. The expression levels of target genes were normalized against endogenous control ACTB. Data analysis was done using 2-ΔΔCt method. Each PCR reaction was performed in triplicate, and the data presented were the average of three independent experiment results for all PCR reactions.
Yang Y., Choppavarapu L., Fang K., Naeini A.S., Nosirov B., Li J., Yang K., He Z., Zhou Y., Schiff R., Li R., Hu Y., Wang J, & Jin V.X. (2020). The 3D Genomic Landscape of Differential Response to EGFR/HER2 Inhibition in Endocrine-Resistant Breast Cancer Cells. Biochimica et biophysica acta. Gene regulatory mechanisms, 1863(11), 194631.
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8

Genotyping of HSF1 Gene Variants

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Genotyping was conducted on Real-time PCR (qPCR). Single nucleotide polymorphisms (rs78202224, rs35253356, rs4977219, and rs34404564 of the HSF1 gene) were assessed with the LightCycler 480 Instrument II Real-Time PCR System (Roche Applied Science, Indianapolis, USA) using the TaqMan genotyping assay (Life Technologies, Foster City, CA, USA). The final volume for each reaction was 10.8 μL, containing 5.5 μL TaqMan Genotyping Master Mix, 0.27 μL TaqMan probe mix, 3 μL DNase/RNase-Free distilled water (Bio Basic Inc. CA) and 2 μL genomic DNA. The real-time PCR steps included an initial activation step at 95°C for 10 min, followed by 40 cycles for genomic DNA from blood and 60 cycles for DNA extracted from tissues sample at of 92°C for 15 sec., and 60°C for 1 min. To validate results from real-time PCR, around five percent of assays were repeated.
Almotwaa S., Elrobh M., AbdulKarim H., Alanazi M., Aldaihan S., Shaik J., Arafa M, & Warsy A.S. (2018). Genetic polymorphism and expression of HSF1 gene is significantly associated with breast cancer in Saudi females. PLoS ONE, 13(3), e0193095.
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9

Quantitative Real-Time RT-PCR for Gene Expression

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Total RNAs were prepared using phenol–chloroform extraction methodology. Reverse transcription of total RNAs was performed using the High‐Capacity cDNA synthesis kit (Thermo Fisher Scientific). Real‐time RT–PCR analysis was performed using the SYBR Green I Master Mix (Roche) and primers listed in Appendix Table S1 on a LightCycler 480 Instrument II Real‐Time PCR system (Roche Molecular Diagnostics) for 45 cycles. Murine GapDH gene was used as internal control for quantifying relative gene expression by using the 2−ΔCt method. All analyses were performed with three replicates as described previously (Hu et al, 2012).
Singer D., Thamm K., Zhuang H., Karbanová J., Gao Y., Walker J.V., Jin H., Wu X., Coveney C.R., Marangoni P., Lu D., Grayson P.R., Gulsen T., Liu K.J., Ardu S., Wann A.K., Luo S., Zambon A.C., Jetten A.M., Tredwin C., Klein O.D., Attanasio M., Carmeliet P., Huttner W.B., Corbeil D, & Hu B. (2018). Prominin‐1 controls stem cell activation by orchestrating ciliary dynamics. The EMBO Journal, 38(2), e99845.
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10

Quantifying Gene Expression by qPCR

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CP RNA was collected and transcribed as described for RT-PCR. The cDNA was diluted with nuclease-free water (New England Biolabs). All samples were run in triplicate. qPCR was performed using a LightCycler 480 Instrument II real-time PCR system (Roche LifeScience), using LightCycler 480 SYBR Green I Master Mix (Roche LifeScience, 04707516001). qPCR cycle conditions were 95°C for 5 minutes, followed by 45 cycles of 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 10 seconds. Data are displayed as relative fold change in expression using the 2–ΔΔCT method (29 (link)), relative to the calibrator housekeeping genes GAPDH and Rps18. Data are shown as fold change in each compound of interest in treated WT and treated and untreated homozygous animals relative to the normalized control (untreated WT) animals. Primers were validated by sequencing. Supplemental Table 1 contains primer information.
Hochstetler A.E., Smith H.M., Preston D.C., Reed M.M., Territo P.R., Shim J.W., Fulkerson D, & Blazer-Yost B.L. (2020). TRPV4 antagonists ameliorate ventriculomegaly in a rat model of hydrocephalus. JCI Insight, 5(18), e137646.
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