With the aid of Omega Bio-tek E.Z.N.A. Total RNA kit (Doraville, GA, USA), total RNA was prepared from HCMECs and then subjected to reverse transcription using RevertAid First Strand cDNA Synthesis Kit (Fermentas, Shanghai, China). SYBR ® Green PCR master mix (Bio-Rad Laboratories, Inc.) was adopted to perform PCR reactions on the MX3000p PCR system (Agilent, Santa Clara, CA). The calculation of relative gene expression was achieved by 2 -ΔΔCt method [30] (link). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was adopted for normalization. Primer sequences were as follows: CTRP3 forward: 5'-ATGCTTTGGAGGCAGCT-CAT-3', reverse: 5'-TCACCTTTGTCGCCCTTCTC-3'; FOXO6 forward: 5'-TCTACGACTGGATGGTCCGT-3', reverse: 5'-GGGTCTTCCCTGTCTTTCCG-3'; GAPDH forward: 5'-AATGGGCAGCCGTTAGGAAA-3', reverse: 5'-GCGCCCAATACGACCAAATC-3'.
Mx3000p pcr system
Manufactured by Agilent Technologies
Sourced in United States, Germany
The MX3000p PCR system is a real-time PCR instrument designed for a variety of applications, including gene expression analysis, pathogen detection, and genotyping. The system provides accurate and sensitive quantification of DNA and RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp virus rna mini kit, by Qiagen (2 mentions)
Lightcycler multiplex rna virus master kit, by Roche (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Total rna kit, by Omega Bio-Tek (1 mentions)
Sybr green pcr master mix, by Bio-Rad (1 mentions)
Sybr green qpcr master mix, by Agilent Technologies (1 mentions)
Revertra plus kit, by Toyobo (1 mentions)
Vysis lsi alk dual color, by Abbott (1 mentions)
Bestar sybrgreen qpcr mastermix, by DBI Bioscience (1 mentions)
Trizol kit, by Takara Bio (1 mentions)
Bestar qpcr rt kit, by DBI Bioscience (1 mentions)
Itaq universal sybr green kit, by Bio-Rad (1 mentions)
10 protocols using mx3000p pcr system
1
Quantifying CTRP3 and FOXO6 Expression
2
qRT-PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was extracted from HPAEpiCs by using TRIzol reagent according to the manufacturer's instructions (15596026, Thermo Fisher, Waltham, MA, USA), and then reverse transcribed to cDNA using 5x primeScript RT Master Mixperfect for Real Time PCR (Takara, Tokyo, Japan). qRT-qPCR was performed by using Bestar® SybrGreen qPCR Master Mix (DBI) on an Agilent Stratagene Mx3000P PCR system. The conditions used for qRT-qPCR were as follows: 95°C for 2 min; 40 cycles of 95°C for 20 s, 58°C for 20 s, and 72°C for 20 s. The 2−△△ct method was used to calculate the relative expression levels of the target gene. GAPDH served as a reference.
3
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Complementary deoxyribonucleic acid was produced using ReverTra-Plus™ kit (Toyobo Life Science) in accordance with the manufacturer’s instructions after ribonucleic acid (RNA) was isolated. The amplification was performed in the MX3000p PCR system (Agilent, Santa Clara, CA) using the SYBR Premix Ex Taq kit (Shanghai Biosteel Biotechnology) with β-actin for normalization. The 2-ΔΔCt method was used to analyze the messenger RNA levels (27 (link)).
4
Quantifying Inflammatory Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mRNA expressions of IKK-α, NF-κB, IL-4, IL-6, IL-8, and TNF-α were determined by quantitative real-time PCR. The cells, incubated with or without essential oil of AL, were subsequently treated with TRIzol reagent as recommended by the manufacturer. Total RNA was isolated utilizing the TRIzol total extraction kit. The concentration of mRNA was measured by a microspectrophotometer with the ratio of OD260/OD280 > 1.8 [17 (link)]. All reverse-transcription of total RNA into cDNA was performed using the PrimeScript® RT reagent kit with gDNA Eraser. Real-time qRT-PCR, which consisted of denaturation at 95°C for 3 min and 40 cycles of denaturation for 5 sec at 95°C and annealing for 1 min at 60°C, was performed in a Stratagene Mx3000p PCR system (Agilent, German). Ct values of mRNAs were analyzed by normalizing with the internal control β-actin. The primers for the genes are presented in Table 1 .
5
Genetic Profiling of EGFR and KRAS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mutation profiles of EGFR and KRAS were examined by using a Human EGFR Gene Mutations detection kit (Real-time Fluorescent PCR) and a Human KRAS Gene Mutations detection kit (Beijing ACCB Biotech, Beijing, China), respectively. In total, the tests examined 63 hotspot mutations including 45 in exons 18, 19, 20, and 21 of EGFR, and 12 in exons 2 and 3 of KRAS. Quantitative polymerase chain reaction (PCR) was performed by using an Mx3000 P PCR system (Agilent, Santa Clara, CA, USA) with the following settings: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The results were interpreted per the manufacturers’ instructions. ALK rearrangement was examined via fluorescence in situ hybridization using a break-apart probe for the ALK gene (Vysis LSI ALK Dual Color; Abbott Molecular, Abbott Park, IL, USA).
6
SARS-CoV-2 Detection by qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Severe Acute Respiratory Syndrome Coronavirus 2 genomic RNA was detected by qRT-PCR using SARS-CoV-2-specific primers and probes for the E, N, and RdRP genes (31 ). In brief, total RNA was extracted from 140 μL swab-UTM using the QIAamp Virus RNA mini kit (QIAGEN, Germany) following the manufacturer’s instructions. One-step qRT-PCR was performed in a 20-μL mixture containing 5 μL of extracted RNA with an Mx3000P PCR System (Agilent, United States) and a LightCycler Multiplex RNA Virus Master kit (Roche Diagnostics, Germany). A cycle threshold (Ct) value <40 indicates a positive result (32 (link)). Negative (RNAse-free water) and positive (RNA extracted from hCoV-19/Taiwan/4/2020, EPI_ISL_411927 virus culture fluid) controls were included. The primers, probe, mixture, machine, and thermal cycling conditions are listed in Supplementary Tables 1A–C .
7
Quantifying Circular RNA hsa_circ_0051908 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TRIzol kit (Takara, Dalian, China, 9108Q) was used to extract total RNA from the samples and confirm the integrity and concentration of total RNA before PCR amplification. cDNA was synthesized using the Bestar qPCR RT Kit (DBI Bioscience, Germany, DBI-2220). Real-time PCR was run on the Agilent Stratagene Mx3000P PCR system (Agilent, CA, USA, Mx3000P) using Bestar® SYBR Green qPCR master Mix (DBI Bioscience, DBI2044). The relative expression of hsa_circ_0051908 was calculated using the 2−ΔΔCt method with GAPDH as the internal control. The following primers were used: hsa_circ_0051908 (F: 5′-ATT CCA CTG AGC GTG CCT AC-3′; R: 5′-AAT GTA GGT GCC CTC AAT AGC-3′) and GAPDH (F: 5′-TGT TCG TCA TGG GTG TGA AC-3′; R: 5′-ATG GCA TGG ACT GTG GTC AT-3′).
8
Real-Time PCR for Mycobacterial DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All specimens were examined by real-time PCR (TaqMan assay) for mycobacterial DNA using an Artus M. tuberculosis RG PCR kit (Qiagen, Hilden, Germany) on Mx3000p PCR system (Stratagene-Agilent) according to the manufacturer's instructions. Briefly, the reaction consisted of 13 μL master mixture, 2 μL of MgCl2 solution, and 10 μL of DNA extract. The cycling profile consisted of initial enzyme activation for 10 min at 95°C, followed by 40 cycles of 2-step PCR amplification of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. This was performed as a part of the routine work of the Microbiology Laboratory of the Medical Research Institute, Alexandria University, Egypt.
9
Quantitative Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from sample cells placed in a 6-well plate (6x104 cells/well) with TRIzol® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols and reverse transcribed into cDNA using the RevertAid cDNA Synthesis kit (Beijing Zhijie Fangyuan Technology Co., Ltd.) according to the manufacturer's protocols. PCR reactions were performed using iTaq Universal SYBR Green kit (Bio-Rad Laboratories, Inc.) on the MX3000p PCR system (Agilent Technologies, Inc.) according to the manufacturer's protocols. RT-qPCR was performed at 50˚C for 2 min and 95˚C for 2 min, followed by 40 cycles at 95˚C for 15 sec and 60˚C for 1 min. The calculation of relative gene expression was operated with the 2-ΔΔCq (17 (link)). The primer sequences were: DOCK8 forward primer: 5'-GGCTGACAGATGAGGCTGG-3', reverse primer: 5'-TCAAAGTCCACTGGCTCGAC-3'; inducible nitric oxide synthase (iNOS) forward primer: 5'-AGGGCCACCTCTACATTTGC-3', reverse primer: 5'-CCCAAGCCATCATTGGGAGT-3'; CD86 forward primer: 5'-TCAATGGGACTGCATATCTGCC-3', reverse primer: 5'-GCCAAAATACTACCAGCTCACT-3' or GAPDH forward primer: 5'-GCCTCCTCCAATTCAACCCT-3', reverse primer: 5'-CTCGTGGTTCACACCCATCA-3'. GAPDH was designated as a standard internal control for relative gene expression. This experiment was repeated three times.
10
SARS-CoV-2 RNA Genome Detection via qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the detection of the SARS-CoV-2 RNA genome, RNA was extracted from 140 μL nasopharyngeal swab samples in UTM using the QIAamp Virus RNA mini kit (QIAGEN, Germany) by following the manufacturer's procedure. Five microliters of RNA was immediately subjected to SARS-CoV-2 one-step qRT–PCR using the LightCycler Multiplex RNA Virus Master kit (Roche Diagnostics, Mannheim, Germany) in an Mx3000P PCR System (Agilent, USA). The SARS-CoV-2-specific primers and probes for the RdRP, E and N genes for qRT–PCR are outlined in Supplementary Table 1 (28 ). A Ct value <40 was considered a positive result (29 (link)). RNAse-free water and the RNA extracted from hCoV-19/Taiwan/4/2020 (EPI_ISL_411927, an isolate obtained from Taiwan Centers for Disease Control) cell culture supernatant were used as a negative control and positive control, respectively, in the qRT–PCR in the study.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!