Cell culture dishes, plates, centrifuge tubes, and other plastic ware were purchased from BD Biosciences (Lincoln Park, NJ); Dulbecco modified Eagle medium (DMEM), Ham F-12, amphotericin B, and gentamicin were from Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). RNeasy Plus Mini RNA extraction kit from Qiagen (Valencia, CA); Ready-To-Go-Primer First-Strand Beads were from GE Healthcare (Piscataway, NJ); TaqMan gene expression assays and real-time PCR master mix were from Applied Biosystems (Foster City, CA). DCFDA—Cellular Reactive Oxygen Species Detection Assay Kit, rabbit polyclonal antibody against human malondialdehyde (MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE), aconitase-2, glutathione peroxidase-1 (GPX1), and mouse monoclonal antibody against superoxide dismutase-1 (SOD1) were purchased from Abcam (Cambridge, MA). Rabbit polyclonal antibody against human heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2) were from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin antibody was from BioLegend (San Diego, CA). Fluorescein Alexa-Flour 488-conjugated secondary antibodies (donkey anti-rabbit, or goat anti-mouse IgG) were from Molecular Probes (Eugene, OR).
Real time pcr master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Real-time PCR master mix is a premixed solution containing all the necessary components for real-time PCR amplification, including DNA polymerase, nucleotides, and buffers. It is designed to enable efficient and accurate detection and quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (6 mentions)
Revertra ace qpcr rt master mix, by Toyobo (5 mentions)
Gentamicin, by Thermo Fisher Scientific (5 mentions)
Rneasy plus mini rna extraction kit, by Qiagen (5 mentions)
Amphotericin b, by Thermo Fisher Scientific (4 mentions)
Superscript 3, by Thermo Fisher Scientific (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Cell culture dishes, by BD (3 mentions)
Centrifuge tube, by BD (3 mentions)
Ready to go primer first strand beads, by GE Healthcare (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Universal probe library upl, by Roche (3 mentions)
L carnitine, by Merck Group (2 mentions)
Betaine, by Merck Group (2 mentions)
Erythritol, by Merck Group (2 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (2 mentions)
7300 real time system, by Thermo Fisher Scientific (2 mentions)
Isogen 2, by Nippon Gene (2 mentions)
31 protocols using real time pcr master mix
1
Oxidative Stress Biomarkers Analysis
2
Quantitative RT-PCR for Synaptic Markers
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The RNA from whole cell pellets or synaptosome pellets was extracted using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Reverse transcription was performed with Random hexamers and SuperScript-III (Invitrogen). Quantitative real-time PCR was carried out with the Applied Biosystems 7300 real-time systems using real-time PCR Master Mix (SYBR Green). The primer sequences were as follows: synaptophysin sense, 5’-AGACATGGACGTGGTGAATCA-3′; antisense, 5’-ACTCTCCGTCTTGTTGGCAC-3′; GAPDH sense, 5′- AGCAGTCCCGTACACTGGCAAAC-3′; antisense, 5′- TCTGTGGTGATGTAAATGTCCTCT-3′.. Each experiment was performed in triplicate in three independent experiments.
3
Quantitative Real-Time PCR Assay
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Aliquots of the cDNA were used for quantitative PCR with real-time PCR Master Mix (Exiqon using an ABI (Applied Biosystems) apparatus according to the manufacturer’s instructions. Primer pairs were obtained commercially from Parsgenome). 5s rRNA was used as internal control and PCR normalization. qRT-PCR was run under conditions of initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 5 seconds, 63°C for 20 seconds, and 72°C for 30 seconds. All qRT-PCR tests were performed in duplicate.
4
Inflammatory Cytokine Assay Protocol
5
Inhibition of Quorum Sensing by Gnaphalium hypoleucum
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To further explore the possible mechanisms for inhibition of QS by active compounds from Gnaphalium hypoleucum DC. [6 (link)], qRT-PCR was performed to investigate the transcription level of the vioABCDE operon with and without active compounds, Control active compound concentrations of 31.25, 15.63, 7.81 µg/mL were determined, using rpoB (RNA polymerase subunit B) as the reference gene. Total RNA was extracted using a RNA prep Pure Cell/Bacteria Kit, according to the manufacturer’s guidelines. Then, the oligonucleotide primers were designed and synthesized by the Shanghai Shenggong company (Table S1 ). Quantitative RT-qPCR was performed using the Real-Time PCR Master Mix (SYBR Green) and an ABI PRISM 7500 Real-time PCR system (Applied Biosystems) with two independent cultures. All experiments were performed in triplicate. The 2−ΔΔCt method was used to analyze the data of the quantitative real-time PCR.
6
Melanoma Cell Total RNA Extraction and qRT-PCR
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Total RNA from melanoma cells was isolated using TRIzol reagent (Invitrogen). Synthesis of cDNA was carried out with a QuantScript RT Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. Quantitative RT‐PCR was carried out in an ABI 7500 Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA) with real‐time PCR Master Mix (SYBR Green). The PCR primers are listed in Table S2. GAPDH was selected as the endogenous control in this assay, and the 2−ΔΔCt method was used to analyze the relative gene expression data.25
7
Quantitative Real-Time PCR Assay
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Isolated total RNA was reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, U.S.A.). Quantitative real-time polymerase chain reaction (PCR) was performed using Realtime PCR Master Mix (Applied Biosystems) and the Universal Probe Library (UPL; Roche Life Science, Penzberg, Germany) in a final volume of 10 μl. Real-time PCR assays were performed using a CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, U.S.A.).
8
RNA Isolation and RT-qPCR Analysis of Retinal Samples
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The RNA from retinae was extracted with the RNeasy Mini Kit (Qiagen, Germany). Reverse transcription was carried out using Random hexamers and SuperScript-III (Invitrogen). Quantitative real-time PCR was performed with the Applied Biosystems 7300 real-time systems using real-time PCR Master Mix (SYBR Green). The primer sequences are listed in Additional file 2 (Supplementary Table 1 ).
9
Quantitative Analysis of Adipogenic Genes
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Total RNA was reverse-transcribed using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, japan), and the resultant cDNA was mixed with Real-Time PCR Master Mix (Applied Biosystems, Waltham, MA) and matching probes and primers specific for human β-actin, PPARγ, SREBF1, C/EBPα, C/EBPβ, C/EBPδ, KLF15, FABP, DLK1, CD24, NANOG, and Oct3/4 (Applied Bioscience) (Supplementary Table S1 ). Real time PCR was carried out on a Step One Plus Real-Time PCR System (Applied Biosystems). The level (average ± S.D.) was normalized with respect to the β-actin mRNA level in each sample, and is expressed as a value relative to the control group (HDFs).
10
Quantitative RT-PCR for Gene Expression
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RNA was extracted with Trizol reagent according to manufacturer's protocol (Invitrogen, Eugene, OR, USA). One normal prostate RNA was purchased from Clontech (Clontech, Palo Alto, CA). Reverse transcription was performed with Random hexamers and SuperScript-III (Invitrogen, Eugene, OR, USA). Quantitative real-time PCR was carried out with the Applied Biosystems 7300 real-time systems using real-time PCR Master Mix (SYBR Green). The standard curve was used to establish amplification efficiency [45 (link)]. All primers used are listed in Table 3 . Each experiment was performed in triplicate in three independent experiments.
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