Rotor gene q 5plex hrm apparatus

Manufactured by Qiagen

Sourced in Germany

The Rotor Gene Q 5plex HRM apparatus is a real-time PCR system designed for high-resolution melting analysis. It features five separate reaction channels and can perform high-resolution melting experiments to analyze DNA samples.

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Lab products found in correlation

Tb green premix ex taq 2, by Takara Bio (1 mentions) Onescript cdna synthesis kit, by Applied Biological Materials (1 mentions)

2 protocols using rotor gene q 5plex hrm apparatus

Quantitative Analysis of mRNA Expression

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For mRNA gene expression, 1 μg of purified RNA was reverse-transcribed with OneScript® cDNA Synthesis Kit (Applied Biological Materials Inc., Canada) according to the manufacturer’s instructions. qPCR reactions were conducted in a Rotor Gene Q 5plex HRM apparatus (Qiagen, Germany) in a 10 μl total reaction volume using TB Green Premix Ex Taq II (Clontech Laboratories, USA) according to the manufacturer’s instructions. Each reaction was run in triplicate and always included a no-template control. The mRNA expression of the genes of interest was calculated using GAPDH as the reference gene.
mRNA expression levels were analysed by the 2^−ΔCt method. The value of the relative expression of the genes of interest is given as mean ± standard deviation (SD) of three independent experiments.
The primers sequences (written 5ʹ-3ʹ) were: p16, Fw: CATAGATGCCGCGGAAGGT, Rv: CTAAGTTTCCCGAGGTTTCTCAGA; IL-1β, Fw: CCAGCTACGAATCTCCGACC, Rv: TGGGGTGGAAAGGTTTGGA; IL-6, Fw: CCAGCTACGAATCTCCGACC, Rv: CATGGCCACAACAATGACG; IL-8, Fw: TCTGCAGCTCTGTGTGTGAAGG, Rv: TGGGGTGGAAAGGTTTGGA; β-actin, Fw: TGCTATCCCTGTACGCCTCT, Rv: GTGGTGGTGAAGCTGTAGCC; DNMT1, Fw: AGAACGCCTTTAAGCGCCG; Rv: CCGTCCACTGCCACCAAAT; SIRT1, Fw: AGGCCACGGATAGGTCCATA; Rv: GTGGAGGTATTGTTTCCGGC. Primer concentration was 200 nM.

Mensà E., Guescini M., Giuliani A., Bacalini M.G., Ramini D., Corleone G., Ferracin M., Fulgenzi G., Graciotti L., Prattichizzo F., Sorci L., Battistelli M., Monsurrò V., Bonfigli A.R., Cardelli M., Recchioni R., Marcheselli F., Latini S., Maggio S., Fanelli M., Amatori S., Storci G., Ceriello A., Stocchi V., De Luca M., Magnani L., Rippo M.R., Procopio A.D., Sala C., Budimir I., Bassi C., Negrini M., Garagnani P., Franceschi C., Sabbatinelli J., Bonafè M, & Olivieri F. (2020). Small extracellular vesicles deliver miR-21 and miR-217 as pro-senescence effectors to endothelial cells. Journal of Extracellular Vesicles, 9(1), 1725285.

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Quantitative mRNA Analysis of Senescent HUVEC Cells

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Total RNA from young and Senescent HUVECs in normal and high glucose conditions were isolated using Norgen Biotek Kit (cat. no. 37500, Norgen Biotek Corporation, Canada), according to the manufacturer’s instructions. RNA was stored at −80 °C until use. mRNA expression was assessed as previously described [27 (link)]. Briefly 1 μg of purified RNA is reverse-transcribed following the protocol PrimeScript™ RT Reagent Kit with gDNA Eraser (cat. no. RR047A, Takara Bio Inc. USA). Real-time PCR reactions were conducted in a Rotor Gene Q 5plex HRM apparatus (Qiagen, Germany) in a 10 μl total reaction volume using TB Green Premix Ex Taq II (Tli Rnase H Plus, cat. no. RR420A, Takara Bio Inc.) according to the manufacturer’s instructions. The mRNA expression of the genes of interest was calculated using β-actin as reference gene, using the 2^−ΔCt method.
The primers sequences (written 5ʹ-3ʹ) were:
p16, Fw: CATAGATGCCGCGGAAGGT, Rv: CTAAGTTTCCCGAGGTTTCTCAGA;
IL-1β, Fw: CCAGCTACGAATCTCCGACC, Rv: TGGGGTGGAAAGGTTTGGA;
IL-6, Fw: CCAGCTACGAATCTCCGACC, Rv: CATGGCCACAACAATGACG;
IL-8, Fw: TCTGCAGCTCTGTGTGTGAAGG, Rv: TGGGGTGGAAAGGTTTGGA;
IFN1B, Fw: TGCTCTCCTGTTGTGCTTCT; Rv: CATAGATGGTCAATGCGGCG
β-actin, Fw: TGCTATCCCTGTACGCCTCT, Rv: GTGGTGGTGAAGCTGTAGCC;
Final primer concentration was 200 nM.

Ramini D., Giuliani A., Kwiatkowska K.M., Guescini M., Storci G., Mensà E., Recchioni R., Xumerle L., Zago E., Sabbatinelli J., Santi S., Garagnani P., Bonafè M, & Olivieri F. (2024). Replicative senescence and high glucose induce the accrual of self-derived cytosolic nucleic acids in human endothelial cells. Cell Death Discovery, 10, 184.

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