The relative expression was calculated using the difference between the amplification cycle (Ct) of the resistance genes (MDR1, CDR1, ERG11, ERG3) and the reference endogenous control gene (ACT1), per sample. C. tropicalis ATCC-750 was used as the reference strain. Analysis of the results was performed in StepOne PlusTM Real-Time PCR software, applying double-delta Ct equation (2-ΔΔCT) (Applied Biosystems). This same software allowed efficiency calculation of each evaluated reaction.
Stepone plus real time pcr software
Manufactured by Thermo Fisher Scientific
The StepOne Plus Real-Time PCR software is a data analysis software designed for use with the StepOne Plus Real-Time PCR System. The software provides tools for setting up and managing real-time PCR experiments, as well as analyzing the generated data.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Ovation pico wta system v2, by Tecan (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Power sybr green premix, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Promega (1 mentions)
Oligo dt primer, by Promega (1 mentions)
Step one plus real time pcr assay, by Thermo Fisher Scientific (1 mentions)
Improm 2 reverse transcriptase, by Promega (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Taqman reverse transcription regents, by Thermo Fisher Scientific (1 mentions)
Taqman ribosomal rna control reagents vic probe, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Actinomycin d, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
6 protocols using stepone plus real time pcr software
1
Fungal Resistance Gene Expression
2
Fungal Resistance Gene Expression
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The relative expression was calculated using the difference between the amplification cycle (Ct) of the resistance genes (MDR1, CDR1, ERG11, ERG3) and the reference endogenous control gene (ACT1), per sample. C. tropicalis ATCC-750 was used as the reference strain. Analysis of the results was performed in StepOne PlusTM Real-Time PCR software, applying double-delta Ct equation (2-ΔΔCT) (Applied Biosystems). This same software allowed efficiency calculation of each evaluated reaction.
3
Quantifying Metabolic Gene Expression in Isolated Islets
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Total RNA was prepared from isolated islets using the RNeasy Lipid Tissue Mini Kit (Qiagen) with an on-column DNase digestion step to remove genomic DNA. RNA concentration was determined in triplicate using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and quality checked with an Agilent Bioanalyzer. RNA was amplified using the Ovation Pico WTA System V2 (NuGen) kit and reversed transcribed. qPCR was performed using an Applied Biosystems StepOne Plus Real-Time PCR system. Each reaction consisted of 2 ng amplified cDNA, 0.5 μl Taqman probe, 10 μl Taqman Fast Advanced Master Mix, and nuclease-free water (final volume of 15 μl). Transcript levels of metabolic genes and the reference gene Actb quantified using Taqman probes (for probes see Supplementary Table 2 ). The reaction cycle comprised an initial denaturation for 10 min at 95 °C, followed by 40 cycles of 95 °C/15 s and 60 °C/60 s. All reactions were performed in triplicate. The StepOne Plus Real-Time PCR software (Applied Biosystems) was used to measure threshold cycle (Ct) values. Gene expression was determined using the Pfaffl method.
4
Real-time PCR Analysis of Sebocytes and ORS
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Subsequent to treatment with PM10 and 5 μM punicalagin, 1 μM EGCG, or 1 μM resveratrol for 24 h, total RNA was obtained from sebocytes and ORS cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany); cDNA was synthesized from 3 μg total RNA with a cDNA synthesis kit containing ImProm-II reverse transcriptase and oligo-dT primers based on the manufacturer’s protocol (Promega, Madison, WI, USA).
Real-time polymerase chain reaction (PCR) was conducted with a Step One Plus real-time PCR assay (Applied Biosystems, Foster City, CA, USA). All reactions were conducted with Power SYBR Green premix (Applied Biosystems) using 50 ng cDNA and 10 pM primers. PCR primer sequences are summarized inSupplemental material Table 1 . The amplification cycling conditions were as follows: 95°C for 10 min and 40 cycles at 95°C for 15 s and 60°C for 60 s. The PCR products were evaluated using Step One Plus real-time PCR software (Applied Biosystems).
Real-time polymerase chain reaction (PCR) was conducted with a Step One Plus real-time PCR assay (Applied Biosystems, Foster City, CA, USA). All reactions were conducted with Power SYBR Green premix (Applied Biosystems) using 50 ng cDNA and 10 pM primers. PCR primer sequences are summarized in
5
RNA Extraction and Real-Time qPCR Analysis
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Total RNA was extracted from the juice sacs according to the method described by Ma et al. (2022) [43 (link)]. The RNeasy Mini Kit (Qiagen, Germany) was used to clean the extracted total RNA using on-column DNase digestion. The cDNA was synthesized with 2 µg of purified total RNA using a TaqMan Reverse Transcription Regents (Applied Biosystems, USA).
The gene expression was conducted by real-time quantitative PCR according to the method of Ma et al. (2022) [42 (link)]. As an endogenous control, the TaqMan Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems) was used. The real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) on a StepOnePlus™ system (Applied Biosystems). Each reaction contained template cDNA, 900 nM primers, and a 250 nM probe (TableS2 ). The thermal cycling conditions were 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The data obtained from the StepOnePlus™ real-time PCR software (Applied Biosystems) was used to analyze the gene expression. The results were normalized with the results of 18 S ribosomal RNA. The Real-time quantitative RT-PCR was performed in three replicates for each sample.
The gene expression was conducted by real-time quantitative PCR according to the method of Ma et al. (2022) [42 (link)]. As an endogenous control, the TaqMan Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems) was used. The real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) on a StepOnePlus™ system (Applied Biosystems). Each reaction contained template cDNA, 900 nM primers, and a 250 nM probe (Table
6
Measuring Ornithine Decarboxylase Activity
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ODC enzyme activity was determined by a radiometric assay, measuring the amount of 14 CO2 released from each reaction in which L-[1-14 C] ornithine is enzymatically converted to 14 CO2 by ornithine decarboxylase. Collection and analysis were carried out, as described previously (Coleman and Pegg 1998).
RNA stability using Actinomycin D assay and qRT-PCR Cells were grown to 70 % confluency and treated with 10-μg/ml Actinomycin D (Sigma-Aldrich, St. Louis, MO). RNA was harvested using the TRIzol reagent according to the manufacturer's instructions (Invitrogen) at 0, 4, and 8 h post-Actinomycin D treatment. Total RNA was then used in reverse transcription and PCR amplification. qRT-PCR was performed using the Step One Plus Real-Time PCR system (Applied Biosystems), and the following primers: ODC sense, 5′-CGAGAACCATGAGCAGCTTTAC-3′; ODC antisense, 5′-GCATCCTTATCGTCAGAGGAAG-3′ (annealing at 58 °C); Cyclophilin A sense, 5′-GCAGGTCCATCTACGGAGAG-3′; and Cyclophilin A antisense, 5′-CTGGGAACCGTTGTGTTTGG-3′ (annealing at 58 °C). To quantify relative gene expression, the comparative cycle threshold (C t) method was utilized, and the C t values for the gene of interest were normalized to the C t values of Cyclophilin A, and were presented in relation to the untreated (0 h time point) controls. Analysis was performed on the Step One Plus Real-Time PCR software (Applied Biosystems).
RNA stability using Actinomycin D assay and qRT-PCR Cells were grown to 70 % confluency and treated with 10-μg/ml Actinomycin D (Sigma-Aldrich, St. Louis, MO). RNA was harvested using the TRIzol reagent according to the manufacturer's instructions (Invitrogen) at 0, 4, and 8 h post-Actinomycin D treatment. Total RNA was then used in reverse transcription and PCR amplification. qRT-PCR was performed using the Step One Plus Real-Time PCR system (Applied Biosystems), and the following primers: ODC sense, 5′-CGAGAACCATGAGCAGCTTTAC-3′; ODC antisense, 5′-GCATCCTTATCGTCAGAGGAAG-3′ (annealing at 58 °C); Cyclophilin A sense, 5′-GCAGGTCCATCTACGGAGAG-3′; and Cyclophilin A antisense, 5′-CTGGGAACCGTTGTGTTTGG-3′ (annealing at 58 °C). To quantify relative gene expression, the comparative cycle threshold (C t) method was utilized, and the C t values for the gene of interest were normalized to the C t values of Cyclophilin A, and were presented in relation to the untreated (0 h time point) controls. Analysis was performed on the Step One Plus Real-Time PCR software (Applied Biosystems).
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