Total RNA was extracted from WT, ΔVpFSTF1, and VpFSTF1com using the RNAsimple Total RNA kit (Tiangen, Beijing, China). Total RNA concentrations were measured using a spectrophotometer (Nanodrop ND-1000), and quality was determined by agarose gel electrophoresis. The RNA was inoculated at 42°C for 2 min to digest DNA, and cDNA was synthesized with PrimeScript reagent kit (TaKaRa). qRT-PCR was conducted using an ABI Prism 7300 Fast Real-Time PCR System (Applied Biosystems) with SYBR Premix ExTaq (TaKaRa). Reaction Ct values were determined, and expression levels were calculated using 2–ΔΔCt (Livak and Schmittgen, 2001 (link)). For comparison with WT levels, the transcript levels of candidate genes were normalized to that of the V. pyri actin (KUI53217.1) gene. Eachexperiment was repeated at least three times.
Abi prism 7300 fast real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7300 Fast Real-Time PCR System is a laboratory instrument designed for performing real-time polymerase chain reaction (PCR) analysis. It is capable of detecting and quantifying nucleic acid sequences in real-time. The system includes optical components, thermal cycling capabilities, and software for data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq, by Takara Bio (5 mentions)
Rnaiso plus reagent, by Takara Bio (3 mentions)
Rnasimple total rna kit, by Tiangen Biotech (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Primescript reagent kit, by Takara Bio (1 mentions)
Rox reference dye, by Takara Bio (1 mentions)
Sybr green pcr kit, by Thermo Fisher Scientific (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq reagent, by Takara Bio (1 mentions)
Isospin cell tissue rna, by Nippon Gene (1 mentions)
Superprep cell lysis kit for qpcr, by Toyobo (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Fast sybr green master mix kit, by Thermo Fisher Scientific (1 mentions)
Sybr exscript qrt pcr kit, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green pcr kit, by Takara Bio (1 mentions)
Primescript rt reagent, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr exscript rt qpcr kit, by Takara Bio (1 mentions)
12 protocols using abi prism 7300 fast real time pcr system
1
qRT-PCR Analysis of Gene Expression
2
Quantifying Virulence Gene Expression in Vibrio pyri
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qRT-PCR was performed to determine the molecular mechanisms associated with VpFSTF1-mediated regulation of virulence-related genes in V. pyri. WT and mutant total RNA in three biological replicates was extracted, and cDNA was synthesized as described above. The expression levels of DEGs in the WT and mutant strains were quantified using qRT-PCR (an ABI Prism 7300 Fast Real-Time PCR System (Applied Biosystems) with SYBR Premix ExTaq (TaKaRa). Ct values were exported, and levels of gene expression were calculated using 2–ΔΔCt (Livak and Schmittgen, 2001 (link)). For comparison with WT levels, transcript levels of candidate genes were normalized to those of the V. pyri actin gene. The experiment was repeated at least three times.
3
qRT-PCR Analysis of Defense Genes
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Total RNA was extracted from N. benthamiana leaves using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The cDNA was generated using the PrimeScript RT reagent Kit (Takara). Real-time quantitative PCR was performed in 20- µL reactions including 20 ng of cDNA, 0.2 µM gene-specific primers, 0.4 ul ROX Reference Dye, 10 µL of SYBR Premix Ex Taq (Takara), and 6.8 µL of deionized water. PCR was performed on an ABI PRISM 7300 Fast Real-Time PCR System (Applied Biosystems) under the following conditions: 95°C for 30 s, 40 cycles of 95°C for 5 s and 60°C for 31 s to calculate cycle threshold values, followed by a dissociation program of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s to obtain the melt curves. The N. benthamiana EF1α gene was used as the internal reference gene for calculating relative transcript levels. An equal volume of cDNA was used for gene analysis expression of defense-related genes, using specific primers PR1b 5′- GTGGACACTATACTCAGGTG-3′/5′-TCCAACTTGGAATCAAAGGG-3′, PR2b 5′-AGGTGTTTGCTATGGAATGC-3′/5′-TCTGTACCCACCATCTTGC-3′, ERF1 5′-GCTCTTAACGTCGGATGGTC-3′/5′-AGCCAAACCCTAGCTCCATT-3′, LOX 5′-AAAACCTATGCCTCAAGAAC-3′/5′-ACTGCTGCATAGGCTTTGG-3′ and EF1α 5′-AGAGGCCCTCAGACAAAC-3′/5′-TAGGTCCAAAGGTCACAA-3′. The induction ratio of treatment/control was then calculated by the 2-ΔΔCT approach. Student t-test was used for statistical analysis.
4
Quantitative Analysis of NKAP mRNA Expression
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The messenger RNA (mRNA) expression of NKAP genes was detected by quantitative real-time PCR. Total RNA was extracted from cells and tissues using Trizol reagent and reverse-transcribed into complementary DNA using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, United States). NKAP gene expression was examined using quantitative real-time PCR through an ABI Prism 7300 Fast Real-Time PCR System (Applied Biosystems, United States) and SYBR Green PCR kit (Thermo Fisher Scientific, United States). The primer sequences were as follows: NKAP, forward, 5′ CCGAAGCCCAGCAAATC 3′ and reverse, 5′ AGGAGGCAG AAGCGAAGG 3′; glyceraldehyde-3-phosphate dehydrogenase, forward, 5′ AATCCCATCACCATCTTC 3′ and reverse, 5′ AGGCTGTTGTCATACTTC 3′. The results were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase.
5
RT-qPCR Experiments with SYBR Premix
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RT-qPCR experiments were performed on an ABI Prism 7300 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using the SYBR Premix ExTaq reagent (TaKaRa). The primers used for the qPCR are presented in Table S12 . The relative gene expression levels were calculated using comparative threshold cycle method (2−ΔΔCt) and were normalized by actin [60 (link)]. Correlation between RNA-seq and RT-qPCR was calculated based on the fold changes of RT-qPCR and RNA-seq by format in Microsoft EXCEL2010 (Microsoft, Redmond, Washington, DC, USA).
6
Quantitative RT-PCR analysis of P. sojae
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Total RNA was extracted from mycelia, sporangia and infected soybean leaves using the RNAsimple Total RNA Kit (Tiangen, China) following the recommended protocol. Quantitative RT-PCR was performed in 20 μL reactions including 20 ng cDNA, 0.2 μM gene-specific primer for the reference P. sojae TEF1 gene (EU079791), 10 μL SYBR Premix ExTaq (Takara), and 6.8 μL ddH2O. PCR reactions were performed on an ABI PRISM 7300 fast Real-Time PCR System (Applied Biosystems) under the following conditions: 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 31 s to calculate cycle threshold values, followed by a dissociation program of 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s to obtain melt curves. The 7300 System Sequence Detection Software (version 1.4; SDS) was used to obtain relative expression levels for each sample. The transcript levels for test genes were determined according to the function ΔCT = CT (test gene) − CT (reference gene). To compare untreated and treated expression levels, the function ΔΔCT was determined using the equation ΔΔCT = ΔCT (treatment) − ΔCT (control), in which the control was mock-treated P. sojae P6497 mycelia. The induction ratio of treatment/control was calculated using the equation 2−ΔΔ CT.
7
Quantitative RNA Expression Analysis
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Total RNA was isolated from the cells using the SuperPrep Cell Lysis Kit for qPCR (Toyobo, Osaka, Japan) and was isolated from the tissue using ISOSPIN Cell & Tissue RNA (NIPPON GENE, Toyama, Japan). cDNAs were made using M-MLV Reverse Transcriptase (Promega, Tokyo, Japan). mRNA levels were quantified by qPCR using ABI Prism 7300 fast real-time PCR system (Applied Biosystems, Tokyo, Japan) and SYBR Green FAST qPCR master mix (Kapa Biosystems, Woburn, MA). The conditions for real-time PCRs were 40 cycles at 94 ˚C for 15 sec followed by 40 cycles at 60 ˚C for 60 sec. The relative mRNA expression levels were normalized to the levels of HPRT or GAPDH mRNA expression. All primers are listed in Supplementary Table 2.
8
Quantifying Hepatic CLC-2 Expression
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Total RNA (1 μg) from human liver tissues was isolated using the RNeasy Mini kit (Qiagen, CA, USA) and was reverse-transcribed using the SuperScript III First-Strand Synthesis system. QRT-PCR was performed using Fast SYBR ® Green Master Mix Kit (Applied Biosystems, CA, USA) with an ABI Prism 7300 Fast Real-Time PCR system (Applied Biosystems). The ClC-2 mRNA expression was normalized to the mRNA levels of GAPDH. The specific primer sequences used for the amplification were as follows: ClC-2, 5'-CACAGGTGGTGGCATTGTTG-3' and 5'-GGACTTTCACACCCTGTGCT-3'; GAPDH, 5'-GGGCACGAAGGCTCATCATT-3' and 5'-AGAAGGCTGGGGCTCATTTG-3'.
9
Quantitative Analysis of miR-30c Expression
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Total RNA was extracted from different transfected cells using RNA Iso-plus reagent (Takara Bio, China) and then reverse transcribed into cDNA using PrimeScript RT Reagent Kit (Invitrogen) following the manufacturer's protocol. The primers in this study were designed using Primer 5.0 (Primer-E, Ltd., United Kingdom) as follows: miR-30c RT: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTGAG-3′; U6 RT 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGGAAC-3′. With a standard protocol provided by the manufacturer, qRT-PCR was performed in the ABI PRISM 7300 Fast Real-Time PCR System (Ambion, USA) using the SYBR ExScript qRT-PCR Kit (Takara, China), under the following conditions: 95°C for 5 min, 95°C for 10 s, and 40 cycles at 60°C for 30 s. Each reaction was performed in triplicate. The expression levels of miR-30c were normalized to U6. The results of RT-PCR are reported as 2-ΔΔCt.
10
Quantitative RT-PCR analysis of miRNA and EMT genes
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Total RNA was extracted using RNA Iso-plus reagent (Takara Bio). Ultraviolet spectrophotometer was used for measuring RNA concentration and purity. The reverse transcription PCR process was performed using PrimeScript RT Reagent Kit according to the manufacturer’s protocol.16 (link) Primers were designed by Primer 5.0 (Primer-E, Ltd., Plymouth, United Kingdom), (Table 2 ), and the reverse primer for miR-122 and U6 PCR used as internal control was the Uni-miR qPCR Primer offered by the kit.17 (link) The reverse transcription reaction contained 25 μL of total RNA and miRNA PrimeScript RT Enzyme Mix, and the 20 μL real-time PCR (RT-PCR) was performed in the ABI PRISM 7300 Fast Real-Time PCR System (Ambion, Foster City, CA, USA), with the conditions: 95°C for 5 minutes, 95°C for 10 seconds, and 40 cycles at 60°C for 20 seconds. The reaction for Wnt1- and EMT-related genes was performed under conditions of 95°C for 10 seconds, 95°C for 30 seconds, and 40 cycles at 60°C for 30 seconds. The products were separated by electrophoresis on 0.2% agarose gels. The results of RT-PCR were expressed as 2−ΔΔCt.
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