BV2 cells were seeded into six-well plates and treated as indicated. Total RNA was extracted using the Trizol reagent according to the manufacturer’s instructions. RT-PCR were performed using the One-Step RT-PCR System (Applied Biosystems, Foster City, CA, USA). One microgram of RNA template was reverse-transcribed using a Prime Script RT Master Mix Kit (Clontech, Mountain View, CA, USA). RT-PCR was performed using 2 μL of cDNA solution in a 20 μL reaction mixture containing 10 μL of SYBR Premix Ex Taq II, 0.8 μL of the forward primer, 0.8 μL of the reverse primer, and 6 μL ddH2O. Relative mRNA expression was assessed using the comparative ΔΔCt method. Β-actin was used as an internal standard. The RT-PCR primers are shown in Table 1 .
One step rt pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The One-Step RT-PCR System is a laboratory instrument designed for the rapid and efficient amplification of RNA samples. It combines reverse transcription and PCR amplification in a single step, streamlining the process of gene expression analysis and viral detection.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (2 mentions)
Trizol ls, by Thermo Fisher Scientific (2 mentions)
Superscript 3 with platinum, by Thermo Fisher Scientific (2 mentions)
Protein a g bead, by Santa Cruz Biotechnology (2 mentions)
Dntp mix, by Thermo Fisher Scientific (2 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
Qiaamp minelute virus spin kit, by Qiagen (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Hmox1, by Thermo Fisher Scientific (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
Quantstudio 3, by Thermo Fisher Scientific (1 mentions)
Quantstudio 5, by Thermo Fisher Scientific (1 mentions)
Sybr green dye, by Thermo Fisher Scientific (1 mentions)
Lightcycler480 2 sequence detection system, by Roche (1 mentions)
Sybr green real time pcr master mix, by Thermo Fisher Scientific (1 mentions)
α 32p utp, by PerkinElmer (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter system, by Promega (1 mentions)
Human monocyte nucleofector kit, by Lonza (1 mentions)
21 protocols using one step rt pcr system
1
BV2 Cell RNA Extraction and RT-PCR Analysis
2
Quantitative Analysis of Gene Expression
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Total RNA was extracted from brain tissues in each group using the TRIzol reagent, according to the standard instructions. Reverse transcriptase and quantitative PCR were performed on One-Step RT-PCR System (Applied Biosystems). The total RNA template (500 ng) was reverse-transcribed into cDNA using a PrimeScript RT Master Mix kit. Then, 2 ml of cDNA solution was turned into real-time polymerase chain reaction in a 20 μl reaction volume with 10 μl SYBR Premix Ex Taq II, 0.8 μl PCR forward primer (0.4 mM), 0.8 μl PCR reverse primer (0.4 mM), and 6 μl ddH2O. Triplicate reactions were done for every sample. The primers of genes designed for the study are shown in Table 1 .
3
Viral RNA Extraction and Genetic Profiling
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Viral RNA was extracted from 200 µL of each plasma sample using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) according to manufacturer recommendations. cDNA synthesis and the first round of PCR of the NS3, NS5A and NS5B genes were performed using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). Next, a nested PCR was performed using Platinum Taq DNA Polymerase (Thermo Fisher Scientific). The reaction conditions were as follows: 50°C for 30 minutes; 94°C for 2 minutes; 40 cycles at 94°C for 15 seconds, primer annealing for 30 seconds and 68°C for 3 minutes, and 68°C for 5 minutes. The annealing temperatures of NS3, NS5A and NS5B primers are shown in Table 1 .23 (link)–25 (link) Positive and negative controls were always included to control for possible contaminations.
4
RT-qPCR Analysis of Gene Expression
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Total RNA was isolated from the HS181 cell line using the miRNeasy Micro Kit (Qiagen, 217084). We used the One-Step RT-PCR System (Thermo Fisher Scientific, 12574026) with the Taqman Assays IKBKG (Hs00415849_m1, 4453320), METAP1D (Hs00994998_m1, 4448892), HMOX1 (Hs01110250_m1, 4453320), and EIF4B (Hs00973573_m1, 4331182) (all Thermo Fisher). Gene expression was quantified from the isolated RNA at 100 ng/reaction using the Quantstudio real-time qPCR instrument.
5
Quantitative RT-PCR Assay for HIF-1α, VEGF, and GAPDH
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Primers and the TaqMan probes for HIF-1α, VEGF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed by Invitrogen TM (Thermo Fisher Scientific Inc.). The sequences of primers and TaqMan probes are shown in Table 1. The reaction was carried on with One-Step RT-PCR System (Thermo Fisher Scientific Inc.) according to the manufacturer instructions. The PCR conditions were: 2 min at 50°C, 30 s at 95°C, 30 s at 65°C with 40 cycles, and then 1 min at 60°C. PCRs and subsequent calculations were performed with QuantStudio TM 3 and QuantStudio TM 5 (Thermo Fisher Scientific Inc.).
6
Molecular Cloning of Influenza A/Indonesia/5/05 (H5N1) Genes
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Example 22
RT-PCR and Cloning of HA, NA, and M1 Genes of Influenza A/Indonesia/5/05 (H5N1) Virus
Clade 2 influenza virus, strain A/Indonesia/5/05 (H5N1) viral RNA was extracted using Trizol LS (Invitrogen, Carlsbad, Calif.) under BSL-3 containment conditions. Reverse transcription (RT) and PCR were performed on extracted viral RNA using the One-Step RT-PCR system (Invitrogen) with gene-specific oligonucleotide primers. The following primer pairs were used for the synthesis of the H5N1 hemagglutinin (HA), neuraminidase (NA), and matrix (M1) genes, respectively:
Following RT-PCR, cDNA fragments containing influenza HA, NA, and M1 genes with molecular weights of 1.7, 1.4, and 0.7 kB, respectively, were cloned into the pCR2.1-TOPO vector (Invitrogen). The nucleotide sequences of the HA, NA, and M1 genes were determined by DNA sequencing. A similar strategy was followed for cloning a clade 1 H5N1 influenza virus from Vietnam/1203/2003.
7
Quantitative Gene Expression Analysis
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The treated cells were collected and the total RNA was prepared using TRIzol® reagent (Invitrogen Life Technologies). Subsequently, reverse transcription was performed with 2 µg total RNA using a one-step RT-PCR system (Invitrogen Life Technologies). qPCR was conducted in an Applied Biosystems 7300 Real-time PCR Instrument (Applied Biosystems Life Technologies, Foster City, CA, USA). The PCR cycling conditions were as follows: 95°C for 3 min, followed by cycles at 95°C for 10 sec and 60°C for 20 sec, then 72°C for 15 sec. The PCR products were detected using SYBR® Green dye (Applied Biosystems Life Technologies) according to the manufacturer's instructions. Specific oligonucleotide primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Relative quantification of the mRNA levels of target genes was performed using the 2−ΔΔCT method, as described previously (19 (link)).
8
RT-PCR Amplification of p65 cDNA
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In RT-PCR assays, the One-Step RT-PCR System (the Superscript III with Platinum Taq Invitrogen, Carlsbad, CA, USA) was used. To amplify the p65 cDNA, primers specific to p65 gene and three-step cycling, were used: 1) pre-denaturation at 55°C for 30 minutes; 2) 40 cycles of PCR amplification for 15-second, denaturing at 94°C, 30-second annealing at 60°C and 1 minute extension at 68°C; and 3) final extension for 5 minutes at 68°C. PCR products, 0.8 kb and 0.2 kb DNA were studied by DNA gel electrophoresis in a 1.5 % agarose gel.
9
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was performed using One-Step RT-PCR System (Invitrogen). SYBR Green Real-time PCR Master Mix (Invitrogen) and Light Cycler 480 II Sequence Detection System (Roche) were used for qRT-PCR, and mRNA levels were normalized to β-actin. The sequences of the primers used for qRT-PCR are listed in Supplementary Table 1.
10
Quantitative Real-Time RT-PCR of E2-Inducible Genes
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One-Step RT-PCR System (Invitrogen) as described previously (14 (link)). The product formation was monitored continuously during the polymerase chain reaction (PCR) using the Sequence Detection System software (ver. 1.7, Applied Biosystems, Foster City, CA, USA). The accumulated PCR products were detected directly by monitoring the increase in SYBR® reporter dye fluorescence. The mRNA expression levels of E2-inducible genes (progesterone receptor [PGR] and trefoil factor 1 [TFF1]) in the MCF-7 cells treated with CPAE, E2, ICI, or a combination of these compounds were compared to those in the control cells at each time point using the comparative cycle threshold (Ct) method. The following primer sequences were used: hPGR, forward, 5′-GAC GTG GAG GGC GCA TAT-3′ and reverse, 5′-GCA GTC CGC TGT CCT TTT CT3′; hTFF1, forward, 5′-CCC TCC CAG TGT GCA AAT A-3′ and reverse, 5′-CTG GAG GGA CGT CGA TGG TA-3′; and hβ-ACTIN, forward, 5′-TGG CAC CCA GCA CAA TGA A-3′ and reverse, 5′-CTA AGT CAT AGT CCG CCT AGA AGC A-3′. The quantity of each transcript was calculated as described in the instrument manual and normalized to the amount of β-ACTIN, which was the housekeeping gene.
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