Quantitative RT‐PCR was performed as described.7 Primers and probe for ABCB5 transcript and control 18s were ready‐made reagents (Pre‐Developed TaqMan Assay Reagents; Applied Biosystems). Part of the ABCB5 expression data had been reported previously.7
Pre developed taqman assay reagents
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pre-Developed TaqMan Assay Reagents are a set of pre-designed and validated molecular assay reagents for detecting and quantifying target sequences. They are designed to provide a standardized, high-performance solution for a variety of gene expression, genotyping, and pathogen detection applications.
Automatically generated - may contain errors
Lab products found in correlation
Dna free, by Thermo Fisher Scientific (5 mentions)
Sybr green, by Toyobo (5 mentions)
Realtime pcr master mix, by Toyobo (5 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (3 mentions)
Oubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Abi prism 7700, by Thermo Fisher Scientific (2 mentions)
Superscript vilo master mix, by Thermo Fisher Scientific (1 mentions)
Taqman fast cells to ct kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Isogen, by Nippon Gene (1 mentions)
User bulletin no 2, by Thermo Fisher Scientific (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Taqman real time pcr, by Thermo Fisher Scientific (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
18 protocols using pre developed taqman assay reagents
1
Quantitative RT-PCR for ABCB5 Expression
2
Quantifying HMGB1 and RAGE Gene Expression
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Total RNAs were isolated using an RNeasy Mini kit (Qiagen, Hilden, Germany). First-strand cDNAs were synthesized from 1 μg of each total RNA using Super Script VILO Master Mix (Life Technologies, CA, USA). Two μl of each cDNA was subjected to real-time RT-PCR using TaqMan Gene Expression Assays (Applied Biosystems, EUA) for HMGB1 and RAGE, and Pre-Developed TaqMan Assay Reagents (Applied Biosystems, CA, USA) for β-actin as an internal control. Two independent measurements were averaged and relative gene expression levels were calculated as a ratio to β-actin expression of each sample.
3
Quantitative Analysis of SULF-2 and Cyclin D1 in RCC
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RNA isolation from clinical RCC samples was performed using ISOGEN (Nippon Gene, Tokyo, Japan) in accordance with the manufacturer's instructions. cDNA was constructed using the SuperScript III First‐strand Synthesis System for RT‐PCR (Invitrogen, Carlsbad, CA, USA). RNA isolation and cDNA construction from human RCC cell lines were performed using the TaqMan Fast Cells‐to‐CT Kit (Ambion/Life Technologies, Carlsbad, CA, USA). Each cell line was plated at a density of 8000 cells/well on a 96‐well culture plate. TaqMan PCR reagents for SULF‐2 (Hs01016476_m1) and cyclin D1 (Hs00765553_m1) were purchased from Applied Biosystems (Foster city, CA, USA). Quantitative real‐time PCR was carried out using the TaqMan Master Mix Reagent Kit protocol with a StepOne real‐time PCR system (Applied Biosystems). The data were standardized against β‐actin gene expression using Pre‐Developed TaqMan Assay reagents (Applied Biosystems). The expression level of SULF‐2 mRNA was determined by the ΔΔCt method based on the normal tissue from one patient as a control. All experiments were conducted at least three times.
4
Quantitative RT-PCR Analysis of Immune Markers
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The cDNA from ~100 ng of starting RNA was used for real-time (RT) PCR using Pre-developed Taqman Assay Reagents (Applied Biosystems, Monza, Italy) on an ABI Prism 7700 thermal cycler (Applied Biosystems) according to the manufacturer's protocol. Reactions were performed in 25 μl volume and each reaction contained the FAM-labeled probe and primers for the given target. Human GAPDH was used as the housekeeping gene. Threshold parameters were maintained constant for GAPDH and for each target throughout the study. Relative quantification was obtained using the same reference sample, cDNA from peripheral blood mononuclear cells from a healthy blood donor, in which all targets were amplifiable, throughout the study, and results were expressed as arbitrary units (a.u.), according to the manufacturer's instructions (User Bulletin no. 2, Applied Biosystems). The following mRNAs have been quantified: chemokine (ccl1, ccl2, ccl3, ccl4, ccl5, ccl20, ccl22 and cxcl10), chemokine receptors (ccr3, ccr4, ccr5, ccr6, ccr7 and cxcr5), cytokines (il1α, il1β, il4, il6, il10, il17, ifnγ, tgfβ and tnfα) and T-reg cell markers (CD25/il2ra and foxp3).
5
Quantification of EPAS1 Gene Expression
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Total RNA was prepared from frozen cell pellets using the QIAGEN RNeasy mini kit (QIAGEN, Inc., Valencia, CA) according to manufacturer instructions. Two micrograms of total RNA extracted from each cell line was reverse-transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). A 1/200 dilution of the cDNA was subjected to real-time RT-PCR using TaqMan Gene Expression Assays (Applied Biosystems) for EPAS1 and Pre-Developed TaqMan Assay Reagents (Applied Biosystems) for ACTB as housekeeping control. Three independent measurements were taken and averaged with relative gene expression levels calculated as ratios over ACTB expression for each cell line.
6
Quantitative Real-time PCR Analysis of Cytokine and Angiogenic Factors
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RNA isolation and cDNA construction were performed using a Cells-to-CTTM Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. TaqMan PCR reagents for IL-6 (Hs00985639) and VEGFA (Hs00900055_m1) were purchased from ABI (Applied Biosystems, Foster, CA, USA). Quantitative Real-time PCR was carried out using TaqMan Master Mix reagents kit protocol with a StepOne Real-time PCR System (Life Technologies). The data were standardized against beta-actin gene expression using Pre-Developed TaqMan Assay Reagents (Applied Biosystems).
7
Laser Capture Microdissection of Kidney Tissues
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Frozen kidney sections were separated into glomeruli and non-glomerulus tissues by laser capture micro-dissection (LM200; Olympus, Tokyo, Japan) as previously described [15] (link). Total RNA was extracted with RNeasy mini kit (Qiagen, Tokyo, Japan). mRNA expression levels were determined by TaqMan real-time PCR (Applied Biosystems, Foster City, CA, USA) [16] (link), [17] (link). Expression levels of all genes were normalized by 18S rRNA (internal control) levels. See Table S2 in File S1 for primer and probe sequences. Eukaryotic 18S rRNA was detected with Pre-Developed TaqMan Assay Reagents (Applied Biosystems).
8
Quantitative Gene Expression Analysis
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Total RNA from cells was extracted by Trizol (Thermo Fisher Scientific) according to the manufacturer’s instruction. cDNAs were synthesized using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) as protocol. Quantification was performed with the ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Primers and probes for GEP were GEP-forward (5′-CAA ATG GCC CAC AAC ACT GA-3′), GEP-reverse (5′-CCC TGA GAC GGT AAA GAT GCA-3′) and GEP-probe (5′-6FAM CCA CTG CTC TGC CGG CCA CTC MGBNFQ-3′). Primer and probe reagents for control 18s were ready-made reagents (Pre-Developed TaqMan Assay Reagents, Applied Biosystems). All experiments were performed in a minimum of three replicates.
9
Quantification of PODXL mRNA Levels
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RNA was extracted from 5x105 cells using the RNeasy Plus Micro Kit from Qiagen according to the manufacturer’s instructions. The concentration and quality of the extracted RNA was assessed using a NanoDrop spectrophotometer (ThermoScientific). The purified RNA was stored at -80°C. Human PODXL mRNA levels were estimated by a two-step qRT-PCR using a customized TaqMan Gene Expression Assay kit (Applied Biosystems) following the manufacturer’s instructions. The human housekeeping gene HuPO (Pre-Developed TaqMan Assay Reagents, Applied Biosystems) was used as the endogenous control and all qRT-PCR reactions were performed in triplicate. qRT-PCR experiments were conducted on an Applied Biosystems 7900 HT Fast Real-Time PCR System, and the Relative Quantitation (Comparative CT) method was used to estimate the relative changes in gene expression using the RQ Manager v.1.2 analysis software (Applied Biosystems).
10
Quantitative Real-Time RT-PCR Protocol
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Total RNA was extracted from tissues using Trireagent (Molecular Research Center, Inc.), after which the RNA was treated with DNA-free (Ambion) to remove contaminating DNA and then subjected to reverse transcription using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time RT-PCR was carried out using an Applied Biosystems 7300 real-time PCR System with SYBR green (Toyobo, Japan) or Realtime PCR Master Mix (Toyobo) and TaqMan probes (MBL). The primers and probes used are listed in Table 1 . Values were normalized to mouse GAPDH (Pre-Developed TaqMan assay reagents, Applied Biosystems).
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