Mx3005p qpcr system

Manufactured by Agilent Technologies

Sourced in United States, Germany, Canada, China, United Kingdom

The Mx3005P qPCR System is a real-time PCR instrument designed for quantitative gene expression analysis. It features a 96-well thermal cycler with a Peltier-based heating and cooling system, and utilizes LED-based excitation and CCD-based detection to provide accurate and sensitive fluorescence measurements. The Mx3005P supports multiple fluorescent dyes and can perform a range of qPCR applications, including gene expression profiling, SNP genotyping, and pathogen detection.

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Mx3005P (177 citations) Stratagene Mx3005P qPCR System (74 citations) Mx3005P system (56 citations) Agilent Mx3005P qPCR system (17 citations) Mx3005P Real-Time QPCR System (17 citations) Mx3005P RT-qPCR system (3 citations) Agilent Stratagene Mx3005P qPCR system (3 citations) Mx3005P QPCR Systems instrument (2 citations) Strategene Mx3005P qPCR system (2 citations) Brilliant III Ultra-Fast QPCR Master Mix on Mx3005P QPCR system (2 citations)

275 protocols using mx3005p qpcr system

Gene Expression Analysis of Chondrocytes in 3D Fibrin Gels

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All gene expression analysis were performed with RNA isolated from fibrin gels (3D). For the isolation of RNA from fibrin gels, the MasterPure Complete RNA Purification Kit (Epicentre, Madison, USA, distributed by Biozym, Hessisch Oldendorf, Germany) was used according to Leyh et al. (11 (link), 13 (link)). The RNA concentration was determined using a NanoDrop Spectrophotometer (260/280nm) (Thermo Fisher Scientific, Waltham, USA). To generate single-stranded cDNA, RNA was reverse transcribed with an AffinityScript QPCR cDNA Synthesis Kit (Stratagene, San Diego, CA, USA) and PCR was performed with the Mx3005P QPCR System from Agilent Technologies using Brilliant II SYBER Green qPCR Mastermix (Agilent Technologies, Santa Clara, CA, USA). Gene expression in chondrocytes were analyzed relatively according to the formula 2^-ΔΔΔCT using the Mx3005P QPCR System (Agilent Technologies, Santa Clara, USA) and were analyzed with the MxPro QPCR software for Mx3000P and Mx3005P QPCR Systems (Stratagene, La Jolla, USA). The results were calibrated to the gene expression in untreated control cells, and normalized to TBP and 18s. Primer sequences are shown in Table 2.

Stöckl S., Eitner A., Bauer R.J., König M., Johnstone B, & Grässel S. (2021). Substance P and Alpha-Calcitonin Gene-Related Peptide Differentially Affect Human Osteoarthritic and Healthy Chondrocytes. Frontiers in Immunology, 12, 722884.

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Quantitative PCR Detection of ASFV

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From blood samples obtained in experiment 1, viral DNA was extracted using a combination of acid phenol extractions and binding nucleic acid to silica particles (as described by [36 (link)]). Extracted DNA samples were tested for the presence of ASFV DNA by quantitative (q) PCR using the Mx3005P qPCR system (Agilent Technologies, Santa Clara, CA, USA), essentially as described by Braae et al. [37 (link)].
For experiment 2, viral DNA was extracted from samples of sera, together with nasal swabs and supernatants of homogenized fecal, vomit and tissue samples using a MagNA Pure LC system (Roche, Basel, Switzerland) with the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche) and the Total NA External Lysis protocol. Viral DNA was extracted from tissue samples obtained from the sows and fetuses using the procedure described previously [36 (link)]. Extracted DNA samples were tested for the presence of ASFV DNA by quantitative (q) PCR using the Mx3005P qPCR system (Agilent Technologies), as described [38 (link)].

Lohse L., Nielsen J., Uttenthal Å., Olesen A.S., Strandbygaard B., Rasmussen T.B., Belsham G.J, & Bøtner A. (2022). Experimental Infections of Pigs with African Swine Fever Virus (Genotype II); Studies in Young Animals and Pregnant Sows. Viruses, 14(7), 1387.

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Quantitative Real-Time PCR Protocol

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Total RNA fraction was isolated from 10⁶ cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA synthesis was performed from 1 μg of total RNA using 200u of SuperScript III Reverse Transcriptase (Invitrogen) primed with random hexamer (Promega), in a 20-μl reaction volume. Quantitative real-time PCRs (qPCRs) were then carried out on duplicates in a final volume of 25 μl containing 2× GoTaq qPCR Master Mix (SYBR Green) from Promega, 0.4 μM each forward and reverse primers and 1 μl cDNA. Quantification was performed with the Mx3005P QPCR Systems and MxPro software (Agilent). The values were normalized to rpl38 expression according to the ΔCt method. The oligonucleotides used for amplification have been previously described (Dornier et al, 2012 (link)).

Eschenbrenner E., Jouannet S., Clay D., Chaker J., Boucheix C., Brou C., Tomlinson M.G., Charrin S, & Rubinstein E. (2019). TspanC8 tetraspanins differentially regulate ADAM10 endocytosis and half-life. Life Science Alliance, 3(1), e201900444.

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Quantifying Gene Expression via siRNA Knockdown

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In cases of siRNA transfection, cells were transfected with 35 nM siRNA (Dharmacon) using RNAiMax (Invitrogen). Total RNA was extracted using NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer’s protocol. cDNA synthesis of 1 µg total RNA was performed using iScript cDNA Synthesis Kit (Biorad) following the manufacturer’s guidelines. Quantitative PCR reactions were performed using the Absolute QPCR SYBR Green Mix (ABGene). qPCR reactions were performed on 96 well qPCR plates (ABGene) using either the Mx3000P or Mx3005P qPCR systems (Agilent). Results were calculated as relative mRNA expression (2^ΔΔCt). Data was obtained from at least three independent experiments and is shown as the mean ± StDev. Primer sequences can be found in Supplementary Data 1.

Schneider P., Miguel Bayo-Fina J., Singh R., Kumar Dhanyamraju P., Holz P., Baier A., Fendrich V., Ramaswamy A., Baumeister S., Martinez E.D, & Lauth M. (2015). Identification of a novel actin-dependent signal transducing module allows for the targeted degradation of GLI1. Nature Communications, 6, 8023.

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Quantitative PCR Amplification Protocol

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Quantitative PCR (QPCR) reactions were conducted on Mx3005P QPCR Systems™ (Agilent Technologies, Santa Clara, USA). Amplifications were performed with Brilliant SYBR® II QPCR Master kit (Agilent Technologies, Santa Clara, USA). Following the manufacturer’s instructions, reactions were carried out in a final volume of 25 μl with 1.5 μl of cDNA and 1 μl of each primer at the optimized concentration. Amplifications were performed as described above for conventional PCR assays. A dissociation step was included for all reactions to confirm single specific PCR product amplification and define the Tm of each amplicon. A negative control (no-template control) was included in each primer assay to check for the formation of primer-dimers.

Puech C., Dedieu L., Chantal I, & Rodrigues V. (2015). Design and evaluation of a unique SYBR Green real-time RT-PCR assay for quantification of five major cytokines in cattle, sheep and goats. BMC Veterinary Research, 11, 65.

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Quantitative Analysis of circRNA and mRNA

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The total RNA from the tissues and cells was extracted with the Trizol reagent (Thermo Scientific, U.S.A.), according to the manufacturer’s guide. The quantity of RNA was detectedby Nanodrop spectrophotometer (Thermo Scientific, U.S.A.). Reverse transcription was done with a Reverse Transcription Kit (TaKaRa, Japan) for circRNA and mRNA according to the protocol. Subsequently, quantitative real-time PCR (qRT-PCR) was performed with the Mx3005P QPCR Systems (Agilent Technologies, Inc., U.S.A.), according to the manufacturer’s instructions. GAPDH and small nuclear U6B were used as an endogenous control for circRNA and miRNA, respectively. The primers were constructed by Biosune (Shanghai, China). The relative quantity of target gene was calculated with the 2^−ΔΔCt methods.

Zhang W.Y., Liu Q.H., Wang T.J., Zhao J., Cheng X.H, & Wang J.S. (2019). CircZFR serves as a prognostic marker to promote bladder cancer progression by regulating miR-377/ZEB2 signaling. Bioscience Reports, 39(12), BSR20192779.

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Quantitative PCR Analysis of Gene Expression

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In cases of siRNA transfection, cells were transfected with 35 nM siRNA (Dharmacon) using RNAiMax (Invitrogen). Total RNA was extracted using NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer's protocol. Complementary DNA (cDNA) synthesis of 1 μg total RNA was performed using iScript cDNA Synthesis Kit (Bio-Rad) following the manufacturer's guidelines. Quantitative PCR (qPCR) reactions were performed using the Absolute QPCR SYBR Green Mix (ABGene). qPCR reactions were performed on 96-well qPCR plates (ABGene) using either the Mx3000P or Mx3005P qPCR systems (Agilent). Results were calculated as relative mRNA expression (2^ΔΔCt). Data were obtained from at least three independent experiments and are shown as the mean±s.d. Primer sequences can be found in Supplementary Data 1.

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Quantitative RNA Expression Analysis

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Total RNA was extracted using the NucleoSpin RNA II kit from Macherey-Nagel according to the manufacturer’s protocol. 1 µg of total RNA was used for cDNA synthesis using the iScript cDNA Synthesis Kit (BioRad). For quantitative PCR reactions the Absolute QPCR SYBR Green Mix (Thermo scientific (Waltham, MA, USA)) was used. qPCR reactions were performed on 96 well plates using either the Mx3000P or Mx3005P qPCR systems (Agilent (Santa Clara, CA, USA)). Relative expression was calculated according to the 2^ΔΔCt- method.

Koeniger A., Brichkina A., Nee I., Dempwolff L., Hupfer A., Galperin I., Finkernagel F., Nist A., Stiewe T., Adhikary T., Diederich W, & Lauth M. (2021). Activation of Cilia-Independent Hedgehog/GLI1 Signaling as a Novel Concept for Neuroblastoma Therapy. Cancers, 13(8), 1908.

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Quantitative Gene Expression Analysis

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Twenty-four hours after the treatment of test compounds and LPS, total RNA was isolated from THP-1 cells incubated in co-culture system using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription of mRNA into cDNA was performed with QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Quantification was performed by a Mx3005P QPCR Systems (Agilent Technologies, Santa Clara, CA, USA) by using GoTaq qPCR Master Mix (Promega, Mannheim, Germany) with specific primers. qRT-PCR reactions were performed initial denaturation at 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The data of each gene was normalized to the housekeeping gene GAPDH.

Lee S.M., Kim N.H., Lee S., Kim Y.N., Heo J.D., Jeong E.J, & Rho J.R. (2019). Deacetylphylloketal, a New Phylloketal Derivative from a Marine Sponge, Genus Phyllospongia, with Potent Anti-Inflammatory Activity in In Vitro Co-Culture Model of Intestine. Marine Drugs, 17(11), 634.

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RNA Extraction, cDNA Synthesis, and qPCR Protocol

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Total RNA was extracted using Trizol (Life Technologies, New York). cDNA synthesis was performed with a M-MLV Reverse Transcriptase kit (Promega, Madison) in accordance with the manufacturer's instructions. RT-PCR was performed with ExTaq (Takara Bio). Real-time PCR was performed with GoTaq^® qPCR Master Mix (Promega, Madison). Primer sequences are listed in Table 1. Signals were detected with Mx3000P and Mx3005P QPCR Systems (Agilent Technologies, Santa Clara, USA).

Han S., Guo J., Liu Y., Zhang Z., He Q., Li P., Zhang M., Sun H., Li R., Li Y., Zeng W., Liu J., Lian L., Gao Y, & Shen L. (2015). Knock out CD44 in reprogrammed liver cancer cell C3A increases CSCs stemness and promotes differentiation. Oncotarget, 6(42), 44452-44465.

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