The total RNA of samples was used for first-strand cDNA synthesis with TransScript One Step gDNA Removal and cDNA Synthesis SuperMix kits for qPCR (TransGen Biotech, Beijing, China). qRT-PCR was performed on a CFX96 multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA) with TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). Two internal control genes, glyceraldehyde-3-dehydrogenase (FfGPD) and Ras-related small GTPase (FfRAS), were used as reference genes [33 (link)]. The primers were designed using Primer Premier 6.0 (Table S2 ). Relative gene expression levels were calculated using 2−∆∆Ct threshold cycle calculation [34 (link)].
Cfx96 multicolor real time pcr detection system
Manufactured by Bio-Rad
Sourced in United States, Japan
The CFX96 multicolor real-time PCR detection system is a laboratory equipment designed for real-time polymerase chain reaction (PCR) analysis. It provides a platform for the detection and quantification of nucleic acid sequences using fluorescent dyes or probes.
Automatically generated - may contain errors
Lab products found in correlation
Itaq universal probes supermix, by Bio-Rad (5 mentions)
Mirneasy serum plasma kit, by Qiagen (5 mentions)
Itaq universal sybr green supermix, by Bio-Rad (5 mentions)
Fastquant rt kit with gdnase, by Tiangen Biotech (2 mentions)
Transstart top green qpcr supermix, by Transgene (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green realtime pcr master mix, by Toyobo (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (1 mentions)
Kapa sybr fast qpcr kit, by Roche (1 mentions)
Trizol protocol, by Thermo Fisher Scientific (1 mentions)
Ultrasybr mixture, by CoWin Biotech (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Icycler iqtm5, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
11 protocols using cfx96 multicolor real time pcr detection system
1
Quantitative RT-PCR for Gene Expression Analysis
2
RNA Extraction and qRT-PCR Analysis of Fungal Samples
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Samples for RNA extraction were prepared as previously described (32 (link)). Briefly, 15 small pieces of mycelial mats (1 to 2 mm2) were grown in liquid Vogel’s medium for 13 h and then treated with or without ketoconazole for 12 h or 24 h. Harvested samples were immediately frozen, and then ground into fine powder in liquid nitrogen. Total RNA extraction was performed according to the standard TRIzol protocol (Invitrogen, Carlsbad, CA, USA). The cDNAs were then synthesized from 2 to 3 μg total RNA using the FastQuant RT kit with gDNase (Tiangen, China) according to the manufacturer's protocol, and diluted to a final volume of 50 μL.
Gene-specific primers for qRT-PCR analysis were designed using online tools PrimerQuest or Primer 6 and listed in Table S1C. The qRT-PCR was performed on a CFX96 multicolor real-time PCR detection system (Bio-Rad, Hercules, CA) with SYBR green detection (SYBR green Realtime PCR Master Mix; TOYOBO, Tokyo, Japan) according to the manufacturer's instructions. At least three independent experiments were carried out and each cDNA sample was analyzed in duplicate. The average threshold cycle (CT) values were used to calculate relative expression levels normalized to expression of β-tubulin using the 2−ΔΔCT method (60 (link)).
Gene-specific primers for qRT-PCR analysis were designed using online tools PrimerQuest or Primer 6 and listed in Table S1C. The qRT-PCR was performed on a CFX96 multicolor real-time PCR detection system (Bio-Rad, Hercules, CA) with SYBR green detection (SYBR green Realtime PCR Master Mix; TOYOBO, Tokyo, Japan) according to the manufacturer's instructions. At least three independent experiments were carried out and each cDNA sample was analyzed in duplicate. The average threshold cycle (CT) values were used to calculate relative expression levels normalized to expression of β-tubulin using the 2−ΔΔCT method (60 (link)).
3
Quantitative RT-PCR Analysis of Genes and miRNAs
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All primers used for qRT-PCR and semi-quantitative RT-PCR are listed in Additional file 3 . Total RNA was isolated using TRIzol reagent (Invitrogen, USA) and treated with DNase I (Fermentas, UK) according to the to manufacturer’s instructions. First-strand cDNA was synthesized using the PrimeScript™ RT-PCR Kit (TaKaRa, Japan). Real-time quantitative RT-PCR was then carried out using the SYBR® Premix Ex Taq™ Kit (TaKaRa, Japan) in a CFX96 multicolor real-time PCR detection system (Bio-Rad, USA). Sl-actin and Cs-actin were used as the internal control genes. For qRT-PCR analysis of Cs-miR393, U6 was used as internal control gene and the first-strand cDNA was synthesized using Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Japan). The qRT-PCR was carried out using the SYBR® Premix Ex Taq™ II Kit (TaKaRa, Japan). Quantification of mRNA and miRNA levels was based on the comparative cycle threshold (CT) method and calculated as 2-ΔΔCT. Analysis was conducted on the data from three independent reactions (technical replicates) using samples from three biological replicates. All CT values were listed in Additional file 7 .
4
Fungal Transcriptional Response to Antifungal Drugs
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Samples for RNA extraction were prepared as previously described with some modifications [33 (link)]. First, conidia were inoculated in the 9 cm petri dish containing liquid Vogel’s medium. After 24 h of stationary incubation at 28 °C in the dark, a mycelial mat on the surface of the medium formed and was then cut into circular pieces (Φ2-mm). About 15 pieces were transferred to 100 mL fresh Vogel’s medium and incubated at 28 °C with shaking at 200 rpm for 24 h. Mycelium was then harvested and freshly frozen in liquid nitrogen for RNA extraction. To assess transcriptional changes under drugs treatment, the samples were added with the antifungal drug (KTC or PoxB) after 12 h of incubation, and after another 12 h, mycelium was harvested for RNA extraction. Total RNA was extracted according to the standard TRIzol protocol (Invitrogen, Carlsbad, CA, USA). The cDNAs were synthesized from total RNA (2 μg) using a cDNA synthesis kit (FastQuant RT Kit (with gDNase), TIANGEN, Beijing, China) following the manufacturer’s protocol. The qRT-PCR analysis was performed on a CFX96 multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA) with SYBR green detection (KAPA SYBR® FAST qPCR Kits; KAPA BIOSYSTEMS, Boston, MA, USA) according to the manufacturer’s instructions. Gene-specific primers were designed using the online tools PrimerQuest or Primer 5 and are listed in Table S2 .
5
Evaluating β-Tubulin Gene Expression in T. pyriformis
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To evaluate the expression of β-tubulin gene, RT-qPCR was performed on T. pyriformis cells isolated from infected guppies and guppies in the control group. Real-time PCR was performed in a CFX96 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, USA) using the UltraSYBR Mixture (Beijing ComWin Biotech) with the 18S rRNA gene as the reference gene. Real-time PCR conditions were as follows: 95 o C for 30 s; followed by 40 cycles at 95 o C for 10 s and 56 o C for 30 s; and the dissolution curve was conducted at 0.5 o C increasing rate for 5 s from 60 o C to 90 o C. The melting curve was analyzed at the end of the ampli cation cycles to con rm PCR product speci city. The ampli cation e ciency (%) for these genes was from 92.5-105.7. The 2 -ΔΔCt method was used to infer gene expression levels as described by Livak and Schmittgen (2001).
6
Quantitative Analysis of Gene Expression
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Total RNA from undifferentiated and differentiated Caco-2 cells, intestinal tissues (IE and PP) was isolated by using TrizolTM reagent. Then, 1 µg of RNA was reverse transcribed into cDNA using the SuperscriptTM III First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Then, 50 ng cDNA was used to perform Quantitative real-time PCR by iQTM5 iCycler and CFX96 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA) with SYBR Green (Invitrogen, Carlsbad, CA, USA). Mouse and human oligonucleotide primers for RT-PCR were purchased from Invitrogen (Carlsbad, CA, USA). Real time polymerase chain reaction (PCR) was carried out with ALPI, Claudin-2, interleukin (IL)-4, IL-6, Monocyte chemoattractant protein-1 (MCP-1), Occludin and Tumor necrosis factor alpha (TNF-α) with human and mouse-specific primers (Supplementary Table S1 ), resulting in a 200 bp fragment. As a reference gene, we used Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) primers, resulting in a 200-bp fragment. PCR was performed with an initial step of denaturation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 20 s. Melt curves were established for the reactions. Normalized fold expression was calculated by using the 2-∆∆Ct method.
7
Serum RNA Extraction and qPCR Analysis
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Serum RNA was extracted using miRNeasy Serum/Plasma Kit (Qiagen, Germany). Since our commen use housekeping genes may change its expression in tumor serum, thus we introduced an external reference, pGL3 6 . The pGL3 (1 ng, approximately 2×10 8 copies) was added to serum samples according to the manufacturer's protocol using an miRNeasy Serum/Plasma Kit (Qiagen, Germany). The extracted serum RNA was reverse transcribed using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c, USA). Forward (F) and reverse (R) primers were synthesized by TSINGKE Biological Technology Company (China), as follows: LOC284454-F, 5′-ATTACAGGTGGCTCAGGTGT-3′, LOC284454-R, 5′-CTTCAGTGTGCCTCCTCAGT-3′; and pGL3-F, 5′-TCCATCTTGCTCCAACACCC-3′, pGL3-R, 5′-TCGTCTTTCCGTGCTCCAAA-3′. The probe sequences were as follows: LOC284454-P, 5′-FAM-CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1-3′ and pGL3-P, 5′-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ1-3′. Conventional SYBR-qPCR was performed using iTaq universal SYBR Green Supermix (Bio-Rad, USA). TaqMan-qPCR was performed using iTaq Universal Probes Supermix (Bio-Rad,USA). All RT-qPCR procedures were performed using a Bio-Rad CFX96 Multicolor Real-time PCR Detection System. TaqMan-qPCR allowed the simultaneous detection of two probes in the same tube (Bio-Rad, USA).
8
Serum RNA Extraction and Quantification
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Serum RNA was extracted using miRNeasy Serum/Plasma Kit (Qiagen, Germany). Since our commen use housekeping genes may change its expression in tumor serum, thus we introduced an external reference, pGL3 5 . The pGL3 (1 ng, approximately 2×10 8 copies) was added to serum samples according to the manufacturer's protocol using an miRNeasy Serum/Plasma Kit (Qiagen, Germany). The extracted serum RNA was reverse transcribed using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c, USA). Forward (F) and reverse (R) primers were synthesized by TSINGKE Biological Technology Company (China), as follows: LOC284454-F, 5′-ATTACAGGTGGCTCAGGTGT-3′, LOC284454-R, 5′-CTTCAGTGTGCCTCCTCAGT-3′; and pGL3-F, 5′-TCCATCTTGCTCCAACACCC-3′, pGL3-R, 5′-TCGTCTTTCCGTGCTCCAAA-3′. The probe sequences were as follows: LOC284454-P, 5′-FAM-CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1-3′ and pGL3-P, 5′-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ1-3′. Conventional SYBR-qPCR was performed using iTaq universal SYBR Green Supermix (Bio-Rad, USA). TaqMan-qPCR was performed using iTaq Universal Probes Supermix (Bio-Rad,USA). All RT-qPCR procedures were performed using a Bio-Rad CFX96 Multicolor Real-time PCR Detection System. TaqMan-qPCR allowed the simultaneous detection of two probes in the same tube (Bio-Rad, USA).
9
Serum RNA Extraction and Quantification
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Serum RNA was extracted using miRNeasy Serum/Plasma Kit(Qiagen,Germany). Since there is no suitable internal housekeeping gene for RT-qPCR, we added an external parameter, pGL3, to the RNA extraction process for removing systematic errors. The pGL3 (1 ng, approximately 2×10 8 copies) was added to serum samples according to the manufacturer's protocol using an miRNeasy Serum/Plasma Kit(Qiagen, Germany). The extracted serum RNA was reverse transcribed using a Revert Aid First Strand cDNA Synthesis Kit(Thermo Fisher Scientific, USA). Forward(F) and reverse(R) primers were synthesized by TSINGKE Biological TechnologyCompany(China), as follows: LOC284454-F, 5′-ATTACAGGTGGCTCAGGTGT-3′, LOC284454-R, 5′-CTTCAGTGTGCCTCCTCAGT-3′; and pGL3-F, 5′-TCCATCTTGCTCCAACACCC-3′, pGL3-R, 5′-TCGTCTTTCCGTGCTCCAAA-3′. The probe sequences were as follows:LOC284454-P,5′-FAM-CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1-3′ and pGL3-P, 5′-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ1-3′. Conventional SYBR-qPCR was performed using iTaq universal SYBR Green Supermix(Bio-Rad, USA). TaqMan-qPCR was performed using iTaq Universal Probes Supermix(Bio-Rad,USA). All RT-qPCR procedures were performed using a Bio-Rad CFX96 Multicolor Realtime PCR Detection System. TaqMan-qPCR allowed the simultaneous detection of two probes in the same tube(Bio-Rad,USA).
10
Serum RNA Extraction and qPCR Analysis
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Serum RNA was extracted using miRNeasy Serum/Plasma Kit (Qiagen, Germany). Since our commen use housekeping genes may change its expression in tumor serum, thus we introduced an external reference, pGL3 6 . The pGL3 (1 ng, approximately 2×10 8 copies) was added to serum samples according to the manufacturer's protocol using an miRNeasy Serum/Plasma Kit (Qiagen, Germany). The extracted serum RNA was reverse transcribed using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c, USA). Forward (F) and reverse (R) primers were synthesized by TSINGKE Biological Technology Company (China), as follows: LOC284454-F, 5′-ATTACAGGTGGCTCAGGTGT-3′, LOC284454-R, 5′-CTTCAGTGTGCCTCCTCAGT-3′; and pGL3-F, 5′-TCCATCTTGCTCCAACACCC-3′, pGL3-R, 5′-TCGTCTTTCCGTGCTCCAAA-3′. The probe sequences were as follows: LOC284454-P, 5′-FAM-CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1-3′ and pGL3-P, 5′-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ1-3′. Conventional SYBR-qPCR was performed using iTaq universal SYBR Green Supermix (Bio-Rad, USA). TaqMan-qPCR was performed using iTaq Universal Probes Supermix (Bio-Rad,USA). All RT-qPCR procedures were performed using a Bio-Rad CFX96 Multicolor Real-time PCR Detection System. TaqMan-qPCR allowed the simultaneous detection of two probes in the same tube (Bio-Rad, USA).
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