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Lightcycler 480 384

Manufactured by Roche
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Sourced in Switzerland, United States

The Roche LightCycler-480-384 is a real-time PCR instrument designed for high-throughput gene expression analysis and DNA quantification. It features a 384-well format and supports various fluorescent detection chemistries.

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Lab products found in correlation

Nanodrop 1000, by Thermo Fisher Scientific (2 mentions) Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions) Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions) Nucleospin rna extraction kit, by Macherey-Nagel (1 mentions) Qbase 2, by CellCarta (1 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Mirneasy kit, by Qiagen (1 mentions) Purelink mirna kit, by Thermo Fisher Scientific (1 mentions) Mir 31, by Thermo Fisher Scientific (1 mentions) Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Snorna202, by Thermo Fisher Scientific (1 mentions) Random hexamers, by Sangon (1 mentions) M mlv transcriptase, by Takara Bio (1 mentions) Trizol, by Takara Bio (1 mentions) Cfx96 real time pcr system, by Bio-Rad (1 mentions) Sybr green master mix, by Yeasen (1 mentions) Ezdnase enzyme, by Thermo Fisher Scientific (1 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Superscript 4 vilo master mix, by Thermo Fisher Scientific (1 mentions)

4 protocols using lightcycler 480 384

1

Quantitative gene expression analysis by RT-qPCR

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Cells resuspended in QIAzol using a miRNeasy Kit and processed according to the manufacturer’s instructions (QIAGEN) or in RA1 lysis buffer using the RNA NucleoSpin extraction kit (Macherey&Nagel). RNA was quantified using a NanoDrop 1000 (Thermo Scientific) and 500–2,000 ng was reverse transcribed with a High-Capacity cDNA Reverse Transcription Kit (Life Technologies). qPCRs were performed using Fast SYBR Green Master Mix (Life Technologies) and run on a Roche LightCycler-480-384. Data processing with qbase+ 2.6 software (Biogazelle) relies on normalization with a minimum of 2 reference genes. RT-qPCR primer sequences are the following: for PRRX1, forward 5’-CAGGACAATGACCAGCTGAACTC-3’ and reverse 5’-TGTGTCCGCTCAAAGACACG-3’; for ACTB, forward 5’- CTGGAACGGTGAAGGTGACA-3’ and reverse 5’-AAGGGACTTCCTGTAACAATGCA-3’; for RPL13A, forward 5’-CCTGGAGGAGAAGAGGAAAGAGA-3’ and reverse 5’-TTGAGGACCTCTGTGTATTTGTCAA-3’; for SDHA, 5’- TGGGAACAAGAGGGCATCTG-3’ and reverse 5’- CCACCACTGCATCAAATTCATG-3’.
Raha M., Wang G., Seidman M.M, & Glazer P.M. (1996). Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant.. Proceedings of the National Academy of Sciences of the United States of America, 93(7), 2941-2946.
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2

Quantifying miRNA Levels in gWAT and Liver

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Small RNA was isolated from 30 mg gWAT and 5 mg liver using the PureLink miRNA Kit (Invitrogen) according to the manufacturer's protocol. miR-31 (000185, Thermo Fisher Scientific, Germany), miR-31* (002495, Thermo Fisher Scientific), and snoRNA202 (001232, Thermo Fisher Scientific) were reverse transcribed individually using 30 ng for gWAT and 150 ng miRNA for liver with the TaqMan miRNA Reverse Transcription Kit according to vendor's instructions (Thermo Fisher Scientific). All qPCRs were performed with the Roche LightCycler® 480/384 (Roche, Switzerland). Relative expression of miRNA levels was evaluated using snoRNA202 as endogenous control.
Gottmann P., Ouni M., Saussenthaler S., Roos J., Stirm L., Jähnert M., Kamitz A., Hallahan N., Jonas W., Fritsche A., Häring H.U., Staiger H., Blüher M., Fischer-Posovszky P., Vogel H, & Schürmann A. (2018). A computational biology approach of a genome-wide screen connected miRNAs to obesity and type 2 diabetes. Molecular Metabolism, 11, 145-159.
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3

Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was extracted from cells or grated tissue in TRIzol (Takara), reverse transcribed with random hexamers (Sangon) and M‐MLV transcriptase (Takara). qRT–PCR was performed to detect the mRNA levels of the indicated genes on a CFX‐96 real‐time PCR system (Bio‐Rad) or LightCycler‐480‐384 (Roche) with SYBR Green Master Mix (Yeasen). qRT–PCR primer sequences are listed in Table EV2. β‐Actin in human and mouse samples, or GAPDH in zebrafish samples were used to normalize gene expression. The “relative” mRNA levels were calculated using the 2−ΔCt method with the normalized gene. The “fold” changes were calculated using the 2−ΔΔCt method with the normalized gene and compared to the control groups including PBS, mock, WT mock, DMSO mock, siNC, Vector, PBS mock, WT siNC mock.
Zheng X., Xiao J., Jiang Q., Zheng L., Liu C., Dong C., Zheng Y., Ni P., Zhang C., Zhang F., Zhong R., Ding H., Wang Q., Qiu Y., Gao M., Ding J., Shen N., Wei B, & Wang H. (2022). AKT2 reduces IFNβ1 production to modulate antiviral responses and systemic lupus erythematosus. The EMBO Journal, 41(6), e108016.
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4

RNA Extraction and qRT-PCR Protocol

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Total RNA was extracted using E.Z.N.A.Total RNA Kit I (Omega Bio-Tech, Norcross, GA, USA) and RNA concentrations were measured using the NanoDrop Spectrophotometer ND-1000 (ThermoFisher Scientific). Only samples with 260/280 ratios > 2 were included in further analysis. To remove DNA contaminants, 1 μg of the purified RNA was treated with ezDNAse enzyme (Thermo Fisher) before being reverse transcribed into cDNA using the SuperScript IV VILO MasterMix (ThermoFisher Scientific). cDNA was diluted 1:10 and amplified by real-time PCR using the PowerUp SYBR Green Master Mix (ThermoFisher Scientific, San Jose, CA, USA) in a LightCycler 480, 384-well plate (Roche) consisting of 2× SYBR, 10 ng cDNA, 1 μM forward and reverse primers in nuclease free H2O (Table 1). All reactions were run in duplicate, including minus RT and no-template controls under the following thermal cycling conditions: 50 °C, 2 min; 95 °C, 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Melt curve analysis was performed to confirm amplification specificity. Ct values were normalized to the housekeeping genes PPIH, B2M or TBP using the second derivative maximum method.
Shu D.Y., Butcher E.R, & Saint-Geniez M. (2021). Suppression of PGC-1α Drives Metabolic Dysfunction in TGFβ2-Induced EMT of Retinal Pigment Epithelial Cells. International Journal of Molecular Sciences, 22(9), 4701.
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