Mx3000 real time pcr detection system

Manufactured by Agilent Technologies

Sourced in United States

The Mx3000 real-time PCR detection system is a laboratory instrument used for amplification and detection of nucleic acid sequences. It is designed to perform real-time polymerase chain reaction (PCR) analysis.

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Lab products found in correlation

Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions) Rnase free dnase, by Promega (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Sybr premix ex taq 2, by Takara Bio (1 mentions) Trizol reagent, by Takara Bio (1 mentions) Bicinchoninic acid protein assay kit, by Beyotime (1 mentions) Iqtm sybr green supermix, by Bio-Rad (1 mentions)

Close spelling variants of this product

Real-time System REAL Detection System MX3000

3 protocols using mx3000 real time pcr detection system

Analysis of Plant Stress Gene Expression

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Total RNA was prepared from 100 mg plant material (leaves, stems and roots) using Trizol Reagent (Life Technologies, USA). Contaminating genomic DNA was removed by incubating the total RNA with RNase-free DNase (Promega) at 37°C for 30 min. First-strand cDNA synthesis was carried out using 1 μg total RNA and the PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) according to the manufacturer's recommendation.
Eash real-time PCR reaction (20 μL) contained 1 μL cDNA. Amplification was performed with SYBR® Premix ExTaq™ II (TaKaRa), following the manufacturer's protocol. The primer sequences (CdDHN4-F/CdDHN4-R, 18S as an internal control) are provided in Table 1. The cycling conditions were 5 s at 95°C, 40 cycles of amplification at 95°C for 5 s, at 59°C for 10 s and extension at 72°C for 30 s. The PCR reactions were run in an Agilent Mx3000 Real Time PCR Detection System. The relative expression was determined using the 2^−ΔΔCT method (Pfaffl, 2001 (link)). Three biological replicates were examined.

Lv A., Fan N., Xie J., Yuan S., An Y, & Zhou P. (2017). Expression of CdDHN4, a Novel YSK2-Type Dehydrin Gene from Bermudagrass, Responses to Drought Stress through the ABA-Dependent Signal Pathway. Frontiers in Plant Science, 8, 748.

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Expression of BMP7 in hucMSCs and Exosomes

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Total RNA of hucMSCs was extracted using the TRIzol reagent (TaKaRa, Tokyo, Japan). Reverse transcription was performed using a reagent kit (TaKaRa) following the manufacturer’s guidelines for synthesis of cDNA. Real-time PCR was performed using a Mx3000 real-time PCR detection system (Agilent). The relative expression levels of the sample mRNA were calculated using the 2^−ΔΔCt method. Total protein was extracted using cellular protein lysates (Solarbio, Peking, China) and the concentration of cell proteins was determined using the bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Proteins were denatured by boiling the samples for 15 min at 100 °C with sodium dodecyl sulfate. The hucMSCs proteins were denatured, separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 3% bovine serum albumin, the PVDF membrane (0.22 µm) was washed with 1× Tris buffered saline with 1% Tween (TBST). The expression of BMP7 was determined in hucMSCs and hucMSC-sEVs with antibodies against BMP7 (dilution ratio = 1:200) and GAPDH (dilution ratio = 1:2000) (SAB, Sioux Falls, South Dakota, USA) using Western blotting.

Zhu D., Sun Z., Wei J., Zhang Y., An W., Lin Y, & Li X. (2024). BMP7-Loaded Human Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Ameliorate Liver Fibrosis by Targeting Activated Hepatic Stellate Cells. International Journal of Nanomedicine, 19, 3475-3495.

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RNA Extraction and Real-Time PCR Analysis

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Cells were lysed in 0.5 ml TRIzol reagent, and 100 μl chloroform–isoamyl alcohol (49:1, v:v) was added to the homogenate. After vortexing for 1 min, the solution was centrifuged at 12,000 rpm for 20 min at 4°C. The RNA was precipitated by the addition of 0.5 ml isopropanol and kept at −80°C for 1 h. RNA was pelleted by centrifuging the solution at 12,000 rpm for 20 min at 4°C. The RNA pellet was rinsed in ice-cold 75% ethanol, air-dried and dissolved in DEPC-treated ddH₂O. The cDNA was synthesized from total RNA using M-MLV reverse transcriptase. Real-time PCR was performed with universal cycling conditions (15 min at 95°C, followed by 40 cycles of 30 s at 95°C, 1 min at 55°C and 30 s at 72°C) using an Mx3000 real-time PCR detection system (Agilent Tech, CA, USA) with IQTM SYBR Green Supermix (Bio-Rad Labs, LA, USA) according to the manufacturer’s instructions. GAPDH was used as an internal standard. Oligonucleotide sequences for primers in this study were as follows: GADPH (forward: 5′-GACCTGACCTGCCGTCTA-3′; reverse: 5′-AGGAGTGGGTGTCGCTGT-3′); CCR3 (forward: 5′- TCCCTCTGCTCGTTATGG-3′; reverse: 5′-GATGCTTGCTCCGCTCAC-3′); IL-5 receptor (IL5R): (forward: 5′-ATTGAAGGAACTCGTCTC-3′; reverse: 5′-CTCTCACTTGAACATCGTA-3′).

Fu C.H., Tsai W.C., Lee T.J., Huang C.C., Chang P.H, & Su Pang J.H. (2016). Simvastatin Inhibits IL-5-Induced Chemotaxis and CCR3 Expression of HL-60-Derived and Human Primary Eosinophils. PLoS ONE, 11(6), e0157186.

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