Genomic DNA from whole blood containing EDTA was isolated by standard techniques. The rs8193036, rs3819024, rs2275913 and rs8193037 IL-17A single nucleotide polymorphisms (SNPs) were genotyped using 5’ exonuclease TaqMan genotyping assays on an ABI Prism 7900HT Fast Real-Time PCR system, according to manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA).
Abi prism 7900ht fast real time pcr system
Manufactured by Thermo Fisher Scientific
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The ABI Prism 7900HT Fast Real-Time PCR System is a high-throughput, quantitative real-time PCR instrument designed for fast and accurate nucleic acid analysis. The system enables real-time monitoring of DNA amplification and provides precise quantification of target sequences.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (22 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (18 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (12 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (12 mentions)
Rneasy mini kit, by Qiagen (9 mentions)
Iscript cdna synthesis kit, by Bio-Rad (8 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (8 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Trisure reagent, by Meridian Bioscience (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (5 mentions)
Sds 2, by Thermo Fisher Scientific (5 mentions)
Qbase, by CellCarta (4 mentions)
Qiaamp dna blood mini kit, by Qiagen (4 mentions)
Taqman 5 nuclease probe method, by Thermo Fisher Scientific (4 mentions)
Spss statistics v21, by IBM (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Sybr green assay, by Thermo Fisher Scientific (4 mentions)
Superscript 3, by Thermo Fisher Scientific (4 mentions)
Sybr green, by Thermo Fisher Scientific (4 mentions)
130 protocols using abi prism 7900ht fast real time pcr system
1
Genotyping IL-17A SNPs from Whole Blood
2
IL10 Gene Promoter SNP Profiling
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The single‐nucleotide polymorphisms (SNPs) for this study consisted of the promoter region of the IL10 (Genbank: ID 3586 ) gene (rs1800896: A‐592 C; rs1800871 A‐819C and rs1800872: A‐1082G. (www.ncbi.nlm.nih.gov/SNP , BUILD135). SNPs with unknown genotype frequency and with <.05 minor allele frequency (MAF) were excluded. SNPs were genotyped using TaqMan assays on an ABI Prism 7900HT Fast Real‐time PCR system (Applied Biosystems).
3
Quantitative Gene Expression Analysis
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Total RNA was isolated from the mucosa of jejunum and ileum using the Trizol Reagent (Invitrogen, USA), and cDNA was synthesized using the Revert Aid First Strand cDNA synthesis kit (Applied Biosystems, Thermo Fisher Scientific, USA). For relative quantification of gene expression, the ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster, CA) was used. Primers were designed using the Primer 3 plus program, and sequences are listed in Table 1 . The reaction system contained 5 μL SYBR® Premix Ex Taq™ (2x), 0.4 μL PCR forward primer (10 μM), 0.4 μL PCR reverse primer (10 μM), 0.2 μL ROX reference dye (50x), 1.0 μL cDNA, and 3 μL sterilized ddH2O. The thermal profile for all reactions was 30 s at 95°C, then 40 cycles of denaturation at 95°C for 5 s, and annealing at 60°C for 30 s. Each reaction was completed with a melting curve analysis to ensure the specificity of the reaction. All the samples were analyzed in duplicate, and the relative amount of each specific transcript was obtained after normalization against the endogenous control β-actin. The relative amounts of target genes were quantified according to the 2−ΔΔCT method [23 (link)].
4
Quantitative RT-PCR Gene Expression Analysis
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Bacteria were grown to mid-logarithmic growth phase (OD600 ~0.4) in LB medium. Total RNA extraction and cDNA synthesis were performed using SV Total-RNA Isolation System and GoScript™ Reverse Transcription System as described by the manufacturer (Promega Corp., Madison, WI). Real-time PCR included 2.5 ng cDNA and 200 nM primers in SYBR Select Master Mix (Applied Biosystems, Foster City, CA) and were run on an ABI Prism 7900HT Fast Real Time PCR System. Constitutively expressed rrsA and rpoD genes that respectively encode 16S ribosomal RNA and RNA polymerase sigma 70 factor were used as an internal control (Kurabayashi et al., 2014 (link)). Primers are listed in Table 2 . Amplification plot and melting curve data are available upon request.
5
Quantitative Analysis of MED15 Expression
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RNA was isolated from cell line pellets using the Total RNA Purification Mini Spin Column Kit (Genaxxon Bioscience GmbH, Ulm, Germany). RNA quantity and quality were analysed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). cDNA was synthesised using 200 ng total RNA and the PrimeScript RT Reagent kit with gDNA Eraser (Takara Bio, Saint-Germain-en-Laye, France). qPCR was performed using 5 ng/µl cDNA, Takara Bio SYBR Premix Ex Taq II with ROX Plus, and 10 pmol/µl forward and reverse primer. The following primer sequences were used: MED15, forward: 5′-TTGAGGATGATGAGCGGCAG-3′, reverse: 5′-GGAGGTCCTTGTCATCCAGC-3′; and β-actin, Forward: 5′-CCAACCGCGAGAAGATGA-3′, reverse: 5′-CCAGAGGCGTACAGGGATAG-3′. PCR was performed on an ABIPrism 7900 HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Data were analysed using Qbase+ (Biogazelle, Ghent, Belgium) with β-actin as reference gene, applying the 2−∆∆Cq algorithm.
6
Quantifying α-Synuclein and Phosphorylation
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Cells were cultured onto glass-slides and fixed with 4% PFA for 20 min. Samples were blocked in PBS 1X containing 5% Normal Horse Serum and 5% bovine albumin (Sigma-Aldrich) for 1h. After washing in PBS 1X, cells were incubated with primary antibodies anti-αSyn (1:250, Abcam); anti-pser129 αSyn, (1:100; Abcam) for 2 h at room temperature, followed by PBS washes and incubation with secondary antibody (anti-mouse Alexa-488, 1:500; Thermo-Fischer) for 1 h at room temperature. Fluorescence intensity of αSyn and αSyn-p-serine-129 was quantified on at least 25 cells from 3 different experiments. Data are expressed as percentage relative to the untreated group (control). The images were obtained using a Leica SP8 confocal microscope, using a ×63 objective lens, 1.4 NA.
Total RNA was extracted using TRIzol Reagent (Life Technologies, Grand Island, NY) and cDNA generated with ThermoScript (Invitrogen). real-time PCR analysis based upon the intercalation of SYBR® Green on an ABI prism 7900 HT Fast Real Time PCR system (PE Applied Biosystems). The expression level of each gene was normalized to GAPDH. Samples were analyzed in triplicate from 5 different mouse ileum specimens.
Total RNA was extracted using TRIzol Reagent (Life Technologies, Grand Island, NY) and cDNA generated with ThermoScript (Invitrogen). real-time PCR analysis based upon the intercalation of SYBR® Green on an ABI prism 7900 HT Fast Real Time PCR system (PE Applied Biosystems). The expression level of each gene was normalized to GAPDH. Samples were analyzed in triplicate from 5 different mouse ileum specimens.
7
qRT-PCR Analysis of Gene Expression
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1 μg RNA was used for cDNA synthesis with Superscript II (Cat.# 18064022, Invitrogen). qPCR analysis was performed on an ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems). The cycling conditions were as follows: 50 °C for 2 min, denaturation at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s, and a combined annealing and extension step at 60 °C for 60 s. The list of TaqMan assays (Termofisher) is provided (Extended data table 5 ). Every point in the plots of data for qRT-PCR analysis represents one individual patient sample or experimental measurement, total sample size is represented by 3-10 points on the graph. The means of each measurement/experiment were calculated and plotted for presentation and for statistical analysis using GraphPad. Two-sided unpaired t test was used, and p-values, s.d. and error bars are shown on the graphs. No RT control were used.
8
Quantitative PCR Analysis of GLE-Treated Cells
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The quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. MDA-MB-231 cells were treated with GLE (0 and 1.0 mg/ml) for 24 h and total RNA was isolated using RNAeasy (Qiagen). The RNA samples were reverse transcribed into cDNA (RT-PCR) using random hexamer primers and the TaqMan reverse transcription kit (Applied Biosystems). The cDNA (100 ng per sample) was subjected to qPCR analysis in quadruplicate using forward and reverse primers, the TaqMan Universal Master Mix, and a probe (10 μl per reaction) in fast optical 96-well plates. The data were analyzed using the ABI Prism 7900 relative quantification (ΔΔCt) study software (Applied Biosystems). We used primers for HRAS, VIL2, S100A4, MCAM, I2PP2A and FN1 genes with the β-actin gene as the internal control (Applied Biosystems). The gene expressions levels were normalized to β-actin and are presented as arbitrary fold changes compared between the control and GLE-treated cells.
9
Quantitative Assessment of Fer1L4 lncRNA
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To determine the lncRNA expression profile of Fer1L4, we performed quantitative real-time PCR (qRT-PCR). Each qPCR was conducted with 5 ng/µl cDNA template and 10 pmol/µl of each forward and reverse primer using SYBR® Premix Ex Taq™ II with ROX Plus (Takara Bio, Saint-Germain-en-Laye, France). PCR experiments were performed on an ABIPrism 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The following primer sequences were used: Fer1L4 (forward ACA-CAG-TCC-TTG-TGG-GTT-CC; reverse CCT-GTC-TCC-TCC-ATC-TCT-CC). Obtained data were analyzed using Qbase + software (Biogazelle, Ghent, Belgium) with ACTB and PPIA as reference genes in the algorithm. Both genes were shown to be suitable reference genes for RCC studies [19 (link), 20 (link)].
10
Real-Time PCR Gene Expression Analysis
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Isolated RNA was reversed transcribed to cDNA with the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Gene expression was analyzed by real-time PCR performed in an ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Gene Expression Assays (Thermo Fisher Scientific) for Adora1 (Mm01308023_m1), Adora2 (Mm00802075_m1) and Tbp (Mm00446971_m1), and Cdhr1 (Mm00499982_m1) and Tbp (Mm01277042_m1). Gene expression was calculated as 2–ΔCt relative to Tbp as the endogenous control.
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