Total RNA was extracted from TRIzol LS-treated serum samples using MagMax 96 Blood RNA Isolation Kit (Ambion) and purified RNA samples were stored at -20°C until assayed. The qRT-PCR assays were performed on samples in triplicate using an ABI PRISM 7900HT Sequence Detection System with RNA UltraSenseTM one-step Kit (Invitrogen) and TaqMan Probe (ABI) in accordance with the manufacturer’s instructions. MARV Musoke-specific primers MARV_GP2_F (5’-TCACTGAAGGGAACATAGCAGCTAT-3’), MARV_GP2_R (5’-TTGCCGCGAGAAAATCATTT-3’), and probe MARV_GP2_P (6FAM–ATTGTCAATAAGACAGTGCAC-MGB) were used in each reaction. At least six serial 10-fold dilutions of quantified MARV genomic RNA were used to generate a standard curve. Quantification of genome copies per volume serum were calculated from the cycle-threshold value obtained for each sample compared using the standard-curve equation. The lower limit of quantification for this analysis was 1.3 x 105 genomic equivalents/mL of serum.
Prism 7900ht sequence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Prism 7900HT Sequence Detection System is a real-time PCR (Polymerase Chain Reaction) instrument designed for quantitative gene expression analysis. The system utilizes fluorescence-based detection to monitor the amplification of target DNA sequences in real-time during the PCR process. The Prism 7900HT offers high-throughput capabilities, with the ability to process up to 384 samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (15 mentions)
Rneasy mini kit, by Qiagen (10 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (9 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (8 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Superscript first strand synthesis kit, by Thermo Fisher Scientific (4 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (4 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Rox plus, by Takara Bio (3 mentions)
Sybr premix ex taq 2, by Takara Bio (3 mentions)
Superscript 2 reverse transcriptase kit, by Thermo Fisher Scientific (3 mentions)
Platinum quantitative pcr supermix udg with rox, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Random primer, by Promega (2 mentions)
64 protocols using prism 7900ht sequence detection system
1
Quantifying Marburg Virus in Serum
2
Real-Time qPCR Gene Expression Analysis
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RNA was extracted from cells using the RNeasy Plus Mini kit (Qiagen). An aliquot of total RNA was reverse transcribed using an oligo(dT) primer. For the thermal cycle reactions, the cDNA template was amplified (ABI PRISM 7900HT Sequence Detection System) with primers shown in Supplemental Table S5B using the Platinum Quantitative PCR SuperMix-UDG with ROX (11743-100, Invitrogen) under the following reaction conditions: 40 cycles of PCR (95 °C for 15 s and 60 °C for 1 min) after an initial denaturation (95 °C for 2 min). Fluorescence was monitored during every PCR cycle at the annealing step. The authenticity and size of the PCR products were confirmed using a melting curve analysis (with software provided by Applied Biosystems) and gel electrophoresis analysis.
3
ChIP-qPCR Evaluation of p53 Binding
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ChIP assays were performed as described [11 (link)] in triplicate. Briefly, after treatment, the cellular material was cross-linked with methanol free 1% formaldehyde (Sigma). Then, cell lysates were sonicated using conditions that yield 200-500 bp DNA fragments using a Bioruptor XL (Diagenode, Denville, NJ). DNA-protein complexes were immunoprecipitated with 1 μg of DO-1 p53-specific monoclonal antibody per condition. Mouse Ig (Santa Cruz Biotechnology) was used as a negative control. qPCR was performed on immunoprecipitated chromatin to determine p53 enrichment occupancy on TLR3 and p21 promoter regions. Amplification of GADPH promoter region was used as a negative control. ChIP primers for TLR3, p21 and GADPH were previously described [23 (link)–24 (link)]. qPCR and melting curve analysis was performed using the SYBR® Green (Invitrogen) dye detection method on the ABI PRISM 7900 HT Sequence Detection System under default conditions. Enrichment in the ChIP samples was calculated as a fraction of the Input (%).
4
Quantitative RT-PCR for Gene Expression
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RNA was extracted from cells using the RNeasy Plus Mini kit (Qiagen). An aliquot of total RNA was reverse transcribed using an oligo (dT) primer. For the thermal cycle reactions, the cDNA template was amplified (ABI PRISM 7900HT Sequence Detection System) with gene-specific primer sets using the Platinum Quantitative PCR SuperMix-UDG with ROX (11743-100, Invitrogen) under the following reaction conditions: 40 cycles of PCR (95°C for 15 s and 60°C for 1 min) after an initial denaturation (95°C for 2 min). Fluorescence was monitored during every PCR cycle at the annealing step. The authenticity and size of the PCR products were confirmed using a melting curve analysis (using software provided by Applied Biosystems) and a gel analysis. mRNA levels were normalized using GAPDH as a housekeeping gene.
5
Quantitative Analysis of RNA Transcripts
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Total RNA was extracted using High Pure RNA Isolation Kit (Roche Diagnostics,
Mannheim, Germany). Reverse transcription was performed with 2 μg of the total RNA
using High-Capacity Reverse Transcription (Life Technologies). Quantitative PCR analysis was
performed using a Life Technologies Prism 7900HT Sequence Detection System. TaqMan probes and
primers for ABCC11/MRP8 were obtained as assay-on-demand gene expression products (assay ID: Hs01090768_m1, Life Technologies). The mRNA of pepsinogen I was analyzed using the following primer set (sense primer, 5'-CCC GTC TTT GAC AAC ATC TG-3'; anti-sense primer, 5'-CGC TGC CAC TCT TGT CAT C-3'). The mRNA of GAPDH was used as an endogenous control (assay ID: Hs99999905_m1, Life Technologies). The thermal cycler conditions were as follows: held for 10 min at 95°C followed by two-step PCR for 45 cycles of 95°C for 15 sec and 60°C for 1 min. All experiments were performed in quadruplicate. The number of transcripts was calculated from a standard curve obtained by plotting the known input of six different concentrations versus the PCR cycle number at which the detected fluorescence intensity reached a fixed value. Amplification data were analyzed with Prism Sequence Detection Software version 2.1 (Life Technologies). For each sample, data were normalized to GAPDH.
Mannheim, Germany). Reverse transcription was performed with 2 μg of the total RNA
using High-Capacity Reverse Transcription (Life Technologies). Quantitative PCR analysis was
performed using a Life Technologies Prism 7900HT Sequence Detection System. TaqMan probes and
primers for ABCC11/MRP8 were obtained as assay-on-demand gene expression products (assay ID: Hs01090768_m1, Life Technologies). The mRNA of pepsinogen I was analyzed using the following primer set (sense primer, 5'-CCC GTC TTT GAC AAC ATC TG-3'; anti-sense primer, 5'-CGC TGC CAC TCT TGT CAT C-3'). The mRNA of GAPDH was used as an endogenous control (assay ID: Hs99999905_m1, Life Technologies). The thermal cycler conditions were as follows: held for 10 min at 95°C followed by two-step PCR for 45 cycles of 95°C for 15 sec and 60°C for 1 min. All experiments were performed in quadruplicate. The number of transcripts was calculated from a standard curve obtained by plotting the known input of six different concentrations versus the PCR cycle number at which the detected fluorescence intensity reached a fixed value. Amplification data were analyzed with Prism Sequence Detection Software version 2.1 (Life Technologies). For each sample, data were normalized to GAPDH.
6
Quantitative Real-Time PCR for Gene Expression
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Total RNA was reverse transcribed using Quantitect® Reverse Transcription kit (Qiagen, Hilden, Germany) with random primers. Quantitative real-time PCR (QRT-PCR) amplifications were performed in triplicate using PerfeCTa® SYBR® Green FastMix® (Quanta Biosystems, Maryland, USA). Primers were designed using Primer3 Input software (version 0.4.0) and generated by IDT Technologies. Primer sequences are listed in Additional file 1 : Table S1. A 750-nM mix of forward and reverse primers was used per reaction. QPCR reactions were performed using a Prism® 7900HT Sequence Detection System (Life Technologies, California, USA) for 40 cycles in accordance with manufacturer’s guidelines. The absolute copy number was calculated from a standard curve and normalised to the housekeeping gene, TATA-binding protein (TBP), as previously described [23 (link)]. Fold-change values were calculated by comparing the normalised copy number of individual samples to the mean of the control samples.
7
Hippocampal Gene Expression Analysis
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RNA obtained from mouse hippocampi was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Cat#4368813, Thermo Fisher, MA, USA) according to the manufacturer’s instructions. 30–40 ng of RNA was used per reaction and yielded 2-3μg/μl of DNA. Gene expression was measured in 3 replicates. The expression of Nlrp3, Interferon-γ, Il-1β, Il-10, Nfκ
β, Il-6, Tnf-α, Hmgb1, Il-18, GSDMD, Claudin-5, Vegfα, Itgam, Thy-1, Cx3cr1, PSD95, Mapk3, MMP9, MSR1, MIP-1α/CCL3 (Supplementary Table 1 ) was measured by Real-Time Quantitative Reverse Transcription PCR (RT qPCR) using Power SYBR Green PCR Master Mix (Cat# 4367659, Thermo Fisher, MA, USA) in an ABI PRISM 7900HT Sequence Detection System at the Icahn School of Medicine qPCR CoRE. Graphs represent fold change in cDNA values with respect to non-exposed controls normalized to hypoxanthine phosphoribosyltransferase (Hprt) internal control using the 2–ΔΔCt method.
β, Il-6, Tnf-α, Hmgb1, Il-18, GSDMD, Claudin-5, Vegfα, Itgam, Thy-1, Cx3cr1, PSD95, Mapk3, MMP9, MSR1, MIP-1α/CCL3 (
8
Mgmt Expression Quantification via qPCR
9
Quantification of TYMP Expression
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Total RNA was prepared from mouse livers using Isogen (Wako Pure Chemical Industries) and RNeasy (QIAGEN). DNase I–treated total RNA (3 μg) was used to synthesize cDNA with SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific). Real-time RT-PCR contained, in a final volume of 50 μl, 0.75 μg of cDNA, 2.5 μl of 20x TaqMan Assay Mix for human TYMP (Assay ID: Hs00157317_m1) or mouse Tymp (Assay ID: Mm01301807_g1), 2.5 μl of 20x TaqMan Assay Mix for ribosomal RNA (4319413E), and 25 μl of 2x TaqMan UMM (all from Thermo Fisher Scientific). The reaction was performed in a 96-well optical reaction plate using the PRISM 7900HT Sequence Detection System (Thermo Fisher Scientific). Expression data were normalized to the ribosomal RNA expression level.
10
RNA Extraction and Gene Expression Analysis from Blood
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RNA was extracted from total blood samples in a randomly selected group of volunteers using a commercial kit (PreAnalytiX, PAX gene, Quiagen/BD Company, Hilden, Germany) and DNA synthesis was performed using a commercial RT First Strand kit. PAX gene tubes avoid RNA degradation during the transport and storage of blood samples. mRNA levels were analyzed by real-time PCR using TaqMan gene expression assays and the Prism 7900HT Sequence detection System (all from Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. All expression analyses were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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