Total bacterial RNA was isolated using TRIZOL (Invitrogen) following the manufacturer's protocol and was treated with DNase I to remove any residual genomic DNA. RNA was further purified using RNeasy RNA Mini Kit (Qiagen). SuperScript Vilo (Life Technologies) was then used to reverse transcribe 1 µg of RNA. Quantitative real-time PCR (qRT-PCR) reactions included 10-fold dilutions of cDNA, HotStarTaq Plus DNA Polymerase (Qiagen), SYBR Green (Life Technologies), 1x PCR buffer, and 0.2 µM of each primer. qRT-PCR primers were designed using PrimerQuest (Integrated DNA Technologies). dsbA primers were 5’-DsbA 5’-GCTGGCGCAGATATGACTAAAG-3’ and 3’-DsbA 5’-GCAGGAGCTATTACTAGGAATGG-3’. RNA polymerase subunit α (FTL_0261) served as the internal control. FTL_0261 primers were 5’-rpoA1 5’-AGATCAGCCAATAGCTACTTTGACA-3’ and 3’-rpoA1 5’-TCGGTTGGTATCGCAGAAAGTATTC-3’. All qRT-PCR reactions were performed in triplicate and samples without reverse transcriptase were used as negative controls to assess genomic DNA contamination. qRT-PCR reactions were performed and analyzed using a CFX96 Real-Time PCR Detection System instrument (Bio-Rad). Relative dsbA mRNA levels (Fig. 1B inset) were calculated based on FTL_0261 mRNA expression and are presented as percent values relative to WT LVS (set to 100%).
Cfx96 real time pcr detection system instrument
Manufactured by Bio-Rad
Sourced in Japan
The CFX96 Real-Time PCR Detection System is a laboratory instrument designed for advanced real-time polymerase chain reaction (PCR) analysis. It provides precise and reliable detection of targeted genetic sequences.
Automatically generated - may contain errors
Lab products found in correlation
Reverse transcription kit, by Tiangen Biotech (2 mentions)
Tb green premix ex taq 2 2 tli rnaseh plus kit, by Takara Bio (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Ssofast evagreen supermix, by Bio-Rad (2 mentions)
Cfx manager software, by Bio-Rad (2 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Superscript vilo, by Thermo Fisher Scientific (1 mentions)
Hotstartaq plus dna polymerase, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primerquest, by Integrated DNA Technologies (1 mentions)
Rneasy rna mini kit, by Qiagen (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Platinum sybr green qpcr supermix udg kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using cfx96 real time pcr detection system instrument
1
Quantitative Real-Time PCR Analysis of dsbA
2
Quantitative gene expression analysis
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Total leaf RNA extraction was performed using a kit (Yueyang Hua, Yueyang, China, Cat:0416-50gk). The first cDNA strand was synthesized using a reverse transcription kit (Tiangen, Beijing, China. Cat:KR118-02). The primer sequences of the related genes were downloaded from the GenBank library of NCBI. The primer design is shown in Table S1 . qRT-PCR analysis was performed using the reverse transcription product cDNA as the template and 18S as the internal reference gene. qRT-PCR was performed using the TB Green Premix Ex Taq II (2×) (Tli RNaseH Plus) kit (TaKaRa, Kyoto, Japan) in the CFX96 Real-Time PCR Detection System instrument (Bio-Rad Laboratories, Hercules, CA, USA). The reaction system was 20 μL, consisting of 9 μL TB Green Premix Ex Taq II (TliRNaseH Plus), 7 μL ddH2O, 2 μL cDNA template and 1 μL forward and reverse primers. The cycling progression was as follows: 95 °C for 3 min; 95 °C for 10 s and 56 °C for 30 s, for a total of 39 cycles. Gene expression change ploidy analysis was calculated using 2−ΔΔCt, and relative mRNA expression levels were normalized using 18S. The qRT-PCR validation of the DEGs is shown in Figure S1 .
3
Gene Expression Analysis by qRT-PCR
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Total RNA was isolated using RNeasy Plus Mini-kit (Qiagen, Hilden, Germany), following manufacturer’s instructions. First strand cDNA was synthesized from 800 ng of total RNA in a 20 μL final volume reaction, using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions. Real-time qRT-PCR was carried out using SsoFast EvaGreen Supermix (Bio-Rad Laboratories) in the CFX96 real-time PCR Detection System instrument (Bio-Rad Laboratories). To confirm product specificity, a melting curve analysis was performed after each amplification. Gene expression was calculated by the ΔΔCt method, using TATA-binding protein (TBP) as house-keeping gene. Bio-Rad CFX Manager software was used for expression data generation. Primer sequences will be provided upon request.
4
Real-Time qRT-PCR Gene Expression Analysis
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First strand cDNA was synthesized from 800 ng of total RNA in a 20 μL final volume reaction, using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s instructions. Primer pairs were designed in-house using the NCBI Primer Designing Tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ), selecting only the primers spanning an exon-exon junction and producing a PCR amplificate with a length between 70 and 150 base pairs. Real-time qRT-PCR was carried out using SsoFast EvaGreen Supermix (Bio-Rad Laboratories) in the CFX96 real-time PCR Detection System instrument (Bio-Rad Laboratories), using standard PCR conditions. All experiments were performed in triplicates and values showing >0.5 Ct variation compared to triplicate means were discarded. Each assay included a blank. To confirm product specificity, a melting curve analysis was performed after each amplification. Relative gene expression was normalized to ACTB, B2M, GAPDH, and RPLP0 expression using the ΔΔC t method. For statistical analysis and expression data generation, the Bio-Rad CFX Manager software was used. Primer sequences will be provided upon request.
5
Leaf Total RNA Extraction and qRT-PCR Analysis
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Leaf total RNA extraction was performed using a kit (Yueyang Hua, China). The first strand of cDNA was synthesized using a reverse transcription kit (Tiangen, China). The primer sequences of the relevant genes were downloaded separately from the GenBank library of NCBI. The primer design is shown in Table 4 . Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using the product cDNA of reverse transcription as a template, and 18S was used as an internal reference gene. The qRT-PCR was tested using the TB Green Premix Ex Taq II (2×) (Tli RNaseH Plus) Kit (TaKaRa, Japan) on a CFX96 Real-Time PCR Detection System instrument (Bio-Rad Laboratories, Hercules, CA, USA). The reaction system was 10 μL, including 5 μL TB Green Premix Ex Taq II (TliRNaseH Plus), 2 μL ddH2O, 1 μL cDNA template, and 1 μL forward and reverse primers, respectively. The reaction procedure was as follows: 95 °C for 3 min; 95 °C 10 s, 56 °C 30 s, a total of 39 cycles. Gene expression change ploidy analysis was calculated using 2−ΔΔCt, and relative mRNA expression levels were normalized using 18S.
6
Quantitative Analysis of miRNA Expression
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Real-time quantitative PCR (qPCR) reactions were performed using Platinum® SYBR® Green qPCR SuperMix-UDG kit (Thermo Fisher Scientific, Waltham MA, USA) on a CFX96™ Real-Time PCR Detection System instrument (Biorad, Hercules CA, USA) with the following program: 2 min at 50 °C, 1 min at 95 °C, and 50 cycles of: 15 s at 95 °C, 15 s at 51 °C, 20 s at 60 °C. Data were analyzed with the CFX Manager software (Biorad, Hercules CA, USA). The relative gene expression of the different miRNAs of interest were standardized using the miRNA cel-mir-39 spike-in control and were calculated using the 2−∆∆Ct method [65 (link)].
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