Zebrafish larvae were exposed to 1 and 2 at EC50 concentration and to a solvent control group (DMSO 0.1%) for 48 h between 3DPF and 5DPF as described above. Eight biological replicates consisting of a pool of 10 zebrafish larvae each were sampled per group. The protocol for RNA extraction, quantification, and further processing for real-time PCR analysis followed the one described in Reference [43 (link)]. Target gene expression (fatty-acid synthase (FASN), sirtuin 1 (SIRT1), peroxisome proliferator activated receptor gamma (PPARγ), microsomal triglyceride transfer protein (MTP) was normalized to the combination of reference genes (beta-2 microglobulin, B2M/ elongation factor 1-alpha, EF1A). A multiple reference gene approach was chosen for normalization of mRNA expression in order to avoid quantification bias [56 (link)]. Real-time PCR was performed using the iQ5 real-time PCR machine (Bio-Rad) and samples were run as described in Reference [57 (link)].
Iq5 real time pcr machine
Manufactured by Bio-Rad
Sourced in United States, China
The IQ5 real-time PCR machine is a laboratory instrument designed for the detection and quantification of nucleic acid sequences using the real-time PCR technique. It is capable of performing quantitative analysis of DNA or RNA samples in a 96-well microplate format.
Automatically generated - may contain errors
Lab products found in correlation
Iq sybr green supermix, by Bio-Rad (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Iscript cdna synthesis kit, by Bio-Rad (6 mentions)
Sybr green, by Takara Bio (5 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Superscript 3, by Thermo Fisher Scientific (3 mentions)
Jmp software, by SAS Institute (3 mentions)
Primescript rtase, by Takara Bio (3 mentions)
E z n a plant rna kit, by Omega Bio-Tek (3 mentions)
Iscript reverse transcription supermix, by Bio-Rad (3 mentions)
Itaq universal sybr green supermix, by Bio-Rad (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Iqtm sybr green supermix, by Bio-Rad (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Taqman predesigned primers, by Thermo Fisher Scientific (2 mentions)
Ecodry premix, by Takara Bio (2 mentions)
Sybr green pcr master mix, by Takara Bio (2 mentions)
52 protocols using iq5 real time pcr machine
1
Zebrafish Lipid Metabolism Gene Expression
2
RNA Extraction and qPCR Analysis
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Total RNA from lung tissue was isolated by TRIZOL reagent (Life Technologies, Pittsburgh, PA) according to the manufacturer’s instructions. cDNA was generated using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA). Quantitative real-time PCR was performed using SYBR Green Supermix (Bio-Rad) and run in a Bio-Rad iQ5 real-time PCR machine. All data were normalized to 18 s values, analyzed using comparative CT method and presented as the fold-increase over WT controls.
3
RNA Extraction and Real-Time PCR Analysis
4
Gene Expression Analysis in Juvenile Tissues
5
Quantification of GLUT3 Expression
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Total RNA was extracted using Trizol (Invitrogen) and purified according to the manufacturer's guidelines. One microgram of RNA was added as a template to reverse transcriptase reactions carried out using the qScript cDNA synthesis kit (Quanta Biosciences). Quantitative real-time PCRs were carried out with the resulting cDNAs using SYBR Green PCR master mix (Quanta Biosciences) using an iQ5 real-time PCR machine (Biorad) with the following primers: 18S-F (5′-CCCGTTGAACCCCATTCGTGA-3′), 18S-R (5′-GCCTCACTAAACCATCCAATCGG-3′), GLUT3-F (5′-CAACTTCATGTCAACTTCTGGCT-3′), and GLUT3-R (5′-CTCAGTGAGAAATGGGACCCT-3′).
6
Quantifying Pluripotency Genes Expression after ESRRB Delivery
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To quantify expression levels of pluripotency-related genes (OCT4, SOX2, NANOG) after ESRRB delivery, real-time RT-PCR was performed. Total RNA was extracted from cells using TRIzol reagent (Invitrogen Carlsbad). First strand cDNA was synthesized with 2 μg of each RNA using a PrimeScript 1st strand cDNA synthesis kit (TakaraBio, Shiga, Japan) according to the manufacturer's instructions. The quantitative real-time RT-PCR was performed using iQTm SYBR Green supermix (Bio-Rad Laboratories) on a Bio-Rad iQ5 real-time PCR machine. The primer sequences for the genes are listed in supplementary table 1 . Each gene was normalized to β-actin as a housekeeping control. The results were analyzed using the delta-delta Ct method with the use of housekeeping genes36 (link).
7
Quantitative RT-PCR Analysis of Grape Genes
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First-strand cDNA was synthesized from 1 mg DNase-treated (TaKaRa Biotechnology) total RNA with the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China). Quantitative real-time RT-PCR was conducted using SYBR green (TaKaRa Biotechnology) on an IQ5 real-time PCR machine (Bio-Rad, CA, USA). Each reaction was carried out in triplicate with a reaction volume of 25 μl. Cycling parameters were 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60 °C for 30 s. For dissociation curve analysis, a program including 95°C for 15 s, followed by a constant increase from 60°C to 95°C, was included after the PCR cycles. The expression of grape ACTIN1 (GenBank Accession number AY680701 ), amplified with primers F (5′ - GAT TCT GGT GAT GGT GTG AGT - 3′) and R (5′ - GAC AAT TTC CCG TTC AGC AGT - 3′), as well as grape GAPDH (GenBank Accession number CB973647 ), amplified with primers F (5′ - TTC TCG TTG AGG GCT ATT CCA - 3′) and R (5′ - CCA CAG ACT TCA TCG GTG ACA - 3′), were used as internal controls. Relative expression levels were analyzed using the IQ5 software and the normalized-expression method.
8
Quantitative PCR Analysis of Gene Expression
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RNA from cultured cells was isolated by Trizol and reverse transcriptase (BioRad iScript cDNA Synthesis Kit) was used to generate cDNA. Quantitative real-time PCR was performed using BioRad iQ SYBR-Green Super Mix on a BioRad iQ5 Real Time PCR machine. Relative expression was calculated using the ΔΔCt method. HPRT-1 was used as an internal control. Primers used for detection can be found in Supplementary Table S1 .
9
Quantitative Gene Expression Analysis in Leaf Tissues
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Total RNA was extracted from the leaf tissues of ‘Qingxiang’ and ‘Xingling’ varieties using an improved CTAB method [52 ]. First-strand cDNA was synthesized from 1 mg DNase-treated total RNA using a mixture of Oligo dT Primer and Random 6 mers (PrimeScript™ RT Master Mix, TaKaRa, Xi’an, Shaanxi, China) and the reaction volume was 10 μL [53 (link)]. Gene-specific primers were designed and shown in Table S1 . To verify the specificity of the primers, 28 genes of the 2 varieties were amplified by PCR, respectively (Figure S1 ). qRT-PCR analysis was conducted using TB green (TB Green™ Premix Ex Taq™ II, Takara) with an IQ5 real-time PCR machine (Bio-Rad, Hercules, CA, USA). Each reaction was carried out in triplicate with a reaction volume of 20 μL. Cycling parameters were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. For dissociation curve analysis, a program including 95 °C for 15 s, followed by a constant increase from 60 °C to 95 °C, which was included after the PCR cycles. The qRT-PCR contained three biological repeats. The 18S-RNA gene [41 (link)] was used as an internal control. The primer sequences were listed in Table S1 . Relative expression levels were analyzed using the IQ5 software and the normalized expression method. The results were evaluated by the 2−ΔΔCT method [54 (link)]. Details of the experimental conditions are provided in Table S2 .
10
Soybean Total RNA Extraction and qRT-PCR
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Total RNA from tissues of soybean plants was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) or RNA isolation kit (Biotech, Beijing, China) according to the manufacturer's instructions. For each sample, 10 μg of total RNA was digested with RNase‐free DNase I (Promega, Madison, WI) to remove genomic DNA contamination. After DNase I treatment, RNA concentration was determined again using a NanoDrop ND‐2000 UV spectrophotometer (Thermo Scientific, Wilmington, DE, USA). First‐strand cDNA was synthesized from 2 μg total RNA using the Superscript III first‐strand synthesis system (Life Technologies, Carlsbad, CA, USA). All cDNA samples were diluted 50‐fold in sterile water for qRT‐PCR. Gene‐specific primers are listed in Table S1 . Soybean GmACTIN (Glyma.15g034000) was used as internal control. qRT‐PCR data were generated using an iQ5 Real Time PCR machine (Bio‐Rad) in 40 cycles (94°C for 30 s, 58°C for 30s and 72°C for 30 s).
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