For qRT-PCR, the mycelia of wild-type and all the mutant strains were harvested at growth stages (48 h and 72 h incubated on PDA). Total RNA was isolated with TRIzol reagent (Biomarker Technologies, Beijing, China), and the first-strand cDNA was synthesized with All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). QRT-PCR was performed with the Thermo Fisher Scientific Real-time PCR System (Finland) using TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). In the quantitative real-time PCR, AF structural gene aflD and regulator gene aflR were amplified by the primer pairs shown in Table 3 , and actin gene was used as the endogenous reference gene. The relative quantification of each transcript was calculated following the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). All qRT-PCR assays were conducted with technical triplicates for each sample, and the experiment was repeated three times.
Real time pcr system
Manufactured by Transgene
Sourced in Finland, China
The Real-time PCR System is a laboratory instrument designed for the amplification and detection of specific DNA sequences in real-time. It is a versatile tool used in various applications such as gene expression analysis, pathogen detection, and nucleic acid quantification.
Automatically generated - may contain errors
Lab products found in correlation
Transstart top green qpcr supermix, by Transgene (4 mentions)
All in one first strand cdna synthesis supermix, by Transgene (1 mentions)
Transscript all in one first strand cdna synthesis supermix, by Transgene (1 mentions)
Transscript all in one first strand cdna synthesis supermix, by Thermo Fisher Scientific (1 mentions)
Dna free kit, by Transgene (1 mentions)
First strand cdna synthesis supermix, by Transgene (1 mentions)
Eastep super, by Promega (1 mentions)
Sybr green pcr master mix, by Transgene (1 mentions)
Cdna synthesis kit, by Transgene (1 mentions)
5 protocols using real time pcr system
1
Quantitative RT-PCR Analysis of Aspergillus Genes
2
qRT-PCR Transcriptomics Protocol
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cDNA was prepared using TransScript® All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). qRT-PCR was performed with the Thermo Fisher Scientific Real-time PCR System using TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China), and the primers were listed in Table S1 . The PCR amplification program was developed by Gao [22 (link)].
3
Quantifying Aflatoxin Biosynthesis Genes
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Both wild-type and ΔdmtA mutant mycelia were harvested at growth stages (36 h and 72 h incubated on YES Agar). RNA molecules were isolated with TRIzol reagent (Biomarker Technologies, Beijing, China) and purified with the DNA-free kit (TransGen Biotech, Beijing, China). TransScript® All-in-One First-Strand cDNA Synthesis SuperMix was used to synthesize the first strand cDNA, and qRT-PCR was performed with the Thermo Fisher Scientific Real-time PCR System (Finland) using TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). In the quantitative real-time PCR, AF aflC, aflK and aflO structural genes and aflR and aflS regulator genes were amplified by the primer pairs shown in Table 2 . As an endogenous control, the β-tubulin gene was amplified with 9F/9R primers. The relative quantification of the transcripts was calculated by the 2−ΔΔCt method39 (link). All qRT-PCR assays were conducted with technical triplicates for each sample, and the experiment was repeated twice.
4
Quantifying Gene Expression in Germinating Seedlings
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Total RNA was extracted from seedlings on the seventh day of germination using Eastep® Super (Promega, Madison, USA). Total RNA from each sample was converted into cDNA by using First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China). The specific forward primers and the universal reverse primer were designed by Primer 5.0 (Table S11 – PCR was performed using TransStart® Top Green qPCR SuperMix (TransGen, Beijing, China) on an ABI7500 Real-time PCR system. The 20 μL reactions contained 2 μL reverse transcription cDNA products, 10 μL qPCR SuperMix, 2 μL primer mix, and supplemented with nuclease-free water to 20 μL. The qRT-PCR were performed as follows: 94°C for 30 s, followed by 40 cycles of 94°C for 5 s and 60°C for 30 s. Each reaction had three technical replicates, and the expression levels were calculated using the 2−ΔΔCt method. UBQ5 was used as the internal standard.
5
Quantitative Analysis of NAD+ Biosynthesis Genes
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Cells were washed with ice-cold PBS and total RNA was extracted from using TIANGEN RNaesy Mini kit (ER501-01). Reverse transcription was performed using the Transgen cDNA synthesis kit (AT311-03). Quantitative real-time PCR (qRT-PCR) was carried out with the Bio-rad real-time PCR system using Transgene SYBR Green PCR master mix (AQ131-02).
Primers were used as follows:
NMNAT2 Q-PCR NS: 5’-GAGGCAGATATGGAGGTGATTG-3’NMNAT2 Q-PCR CAS: 5’-TTTTGTATTTGCGGAGTATTGAGG-3’;NMNAT1 Q-PCR NS: 5’-TGGGTGGAAGTTGATACATGG-3’NMNAT1 Q-PCR CAS: 5’-TCCAGGCCTTTCTAGAGTAGG -3’NRK1 Q-PCR NS: 5’- GACTCTCCGGGATACTTTGATG-3’NRK1 Q-PCR CAS: 5’- CCTCTTCAGATTTTGTTCCATCC -3’NAMPT Q-PCR NS: 5’- GCTGCCACCTTATCTTAGAGTT -3’NAMPT Q-PCR CAS: 5’- CTTGTCAACTTCTGTAGCAAACC -3’NARPT Q-PCR NS: 5’- GTCCTCATCGTAGTCAGCAAC -3’NARPT Q-PCR CAS: 5’- CACCAGCTTATAGACGCCAC -3’NADSYN1 Q-PCR NS: 5’- CAAGATACAGGCTTGGACCAG-3’NADSYN1 Q-PCR CAS: 5’- CCGCTAGGACTTGAAACGAG -3’GAPDH Q-PCR NS: 5’-TGAAGGTCGGAGTCAACGG-3’,GAPDH Q-PCR CAS:5’-AGAGTTAAAAGCAGCCCTGGTG-3’.
Primers were used as follows:
NMNAT2 Q-PCR NS: 5’-GAGGCAGATATGGAGGTGATTG-3’NMNAT2 Q-PCR CAS: 5’-TTTTGTATTTGCGGAGTATTGAGG-3’;NMNAT1 Q-PCR NS: 5’-TGGGTGGAAGTTGATACATGG-3’NMNAT1 Q-PCR CAS: 5’-TCCAGGCCTTTCTAGAGTAGG -3’NRK1 Q-PCR NS: 5’- GACTCTCCGGGATACTTTGATG-3’NRK1 Q-PCR CAS: 5’- CCTCTTCAGATTTTGTTCCATCC -3’NAMPT Q-PCR NS: 5’- GCTGCCACCTTATCTTAGAGTT -3’NAMPT Q-PCR CAS: 5’- CTTGTCAACTTCTGTAGCAAACC -3’NARPT Q-PCR NS: 5’- GTCCTCATCGTAGTCAGCAAC -3’NARPT Q-PCR CAS: 5’- CACCAGCTTATAGACGCCAC -3’NADSYN1 Q-PCR NS: 5’- CAAGATACAGGCTTGGACCAG-3’NADSYN1 Q-PCR CAS: 5’- CCGCTAGGACTTGAAACGAG -3’GAPDH Q-PCR NS: 5’-TGAAGGTCGGAGTCAACGG-3’,GAPDH Q-PCR CAS:5’-AGAGTTAAAAGCAGCCCTGGTG-3’.
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