RNA was isolated from early exponential phase cultures (OD600 0.5±0.05) that were either non-exposed or exposed to 1.5 mM NaAsO2, and harvested at the indicated time-points. RNA samples were treated with the TURBO DNA-free™ kit (Ambion, Cambridge, UK) according to the manufacturer's instructions, and purified by on-column DNAse I digestion using the RNase-Free DNase Set (Qiagen, Hilden, Germany). Total RNA (1 μg) was reverse transcribed with Transcriptor Reverse Transcriptase (Roche). qRT-PCR reactions were performed in the Light Cycler 480 Real-Time PCR System using Light Cycler Fast Start DNA Master SYBR Green I (Roche). Relative standard curves were constructed for each gene, using triplicate serial dilutions of cDNA. The relative expression of the genes was calculated by the relative quantification method with efficiency correction, using the LightCycler Software 4.1 (Roche). Actin (ACT1) was used as a reference gene. All assays were made using biological triplicates. The oligonucleotides used are listed in supplementary material Table S3 (oligonucleotides 14 to 23).
Lightcycler software 4
Manufactured by Roche
Sourced in Germany, Switzerland
The LightCycler software 4.1 is a data analysis software designed for use with the LightCycler instrument, a real-time PCR platform developed by Roche. The software provides tools for the acquisition, analysis, and management of real-time PCR data generated by the LightCycler instrument.
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Lab products found in correlation
Lightcycler 2, by Roche (5 mentions)
Superscript 2, by Thermo Fisher Scientific (4 mentions)
Sybr premix ex taq, by Takara Bio (4 mentions)
Lightcycler 2.0 instrument, by Roche (3 mentions)
Lightcycler 480, by Roche (3 mentions)
Lightcycler 1, by Roche (3 mentions)
Turbo dna free, by Thermo Fisher Scientific (3 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Atlas rt pcr primer sequences, by Takara Bio (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Lightcycler faststart dna master sybr green 1, by Roche (1 mentions)
Transcriptor reverse transcriptase, by Roche (1 mentions)
Sensifast sybr no rox kit, by Meridian Bioscience (1 mentions)
Lightcycler 2.0 carousel based system, by Roche (1 mentions)
Lightcycler taqman master kit, by Roche (1 mentions)
Statistica 13, by TIBCO Software (1 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (1 mentions)
30 protocols using lightcycler software 4
1
Quantifying Gene Expression Changes
2
Quantitative PCR Analysis of Gene Expression
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Quantitative PCR (qPCR) was performed using a SensiFast SYBR No-ROX Kit (Bioline, Eveleigh, Australia) in a Lightcycler 480 (Roche, Millers Point, Australia). Reaction mixtures contained sample cDNA, forward and reverse primers (400 nM) (refer to primer sequences listed in Table 1 ) and SensiFast No-ROX 2x Master Mix. qPCR was conducted with initial UDG activation at 50 °C for 2 min followed by Taq activation at 95 °C for 2 min. Samples then underwent 45 cycles of denaturation at 95 °C for 5 s, annealing at 61 °C for 10 s and extension at 72 °C for 15 s. A melt curve analysis was performed at 95 °C for 5 s, 60 °C for 1 min and acquisition at 97 °C for 30 s. Assessment of thermal melt curves for all qPCR products indicated that primers yielded a single product (Supplementary Figure S1 ). Samples were then cooled to 40 °C for 1 s. Standard curves were generated using serial dilutions of a mixed cDNA sample. Lightcycler Software 4.1 (Roche, Millers Point, Australia) was used to conduct melting curve and relative quantification analyses. Data was normalised to β-actin and presented as a fold change in gene expression relative to control samples.
3
One-Step RT-PCR Quantification Assay
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A one-step RT-PCR was performed using TaqMan with BHQ quencher probe at 125 nM and 250 nM of primers in a final volume of 20 μL. Five microliters of the extracted RNA was combined with 15 μl of the master mix and the reverse transcription step was performed 95°C for 15 minutes, 60 cycles of 15 seconds at 95°C and 45 seconds at 60°C. All the procedure was performed in Light Cycler® 2.0 Instrument and data was analyzed with the LightCycler® Software 4.1 (Roche Diagnostic, Deutschland-Mannheim, Germany). The primers and the probe used are shown in Table 1 [19 (link)–21 (link)].
4
Gene Expression Analysis in Lilium
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The expression of LlACS (GenBank acc. no KF573522), LlACO (GenBank acc. no KF573523), LlZEP, LlIDL (GenBank acc. no KT716180), LlHSL, and LlMPK6 genes was analyzed in relation to LlACT (GenBank acc. no KP257588), which was used as a reference endogenous control for normalization purposes. RT-qPCR was performed with a LightCycler 2.0 Carousel-Based System (ROCHE Diagnostics GmbH, Germany) and a LightCyclerTaqMan Master Kit (ROCHE Diagnostics GmbH, Germany), using primers and UPL probes designed previously [5 (link),7 (link)]. The list of gene-specific and reference primers used is presented in Supplementary Figure S4 . The qPCR mixture preparation and conditions were as described previously [7 (link)]. Reaction efficiencies (>99%) were calculated based on the standard curves from serial dilutions of cDNA templates, and relative expression values were obtained by LightCycler Software 4.1 (Roche Diagnostics GmbH, Mannheim, Germany). The data are presented as means ± standard deviation (SD) of three biological repeats obtained from three independent experiments.
5
qPT-PCR Data Analysis Protocol
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All presented data are the mean ± standard deviation (SD) from the number of experimental replications indicated in the Figure legends. qPT-PCR data were analysed by the ΔΔCT method using LightCycler software 4.1 (Roche, Basel, Switzerland). To compare the results to the controls, a one-sample t-test (p < 0.05) was used. For more than two groups of data, one-way ANOVA and Duncan’s post hoc analysis (p < 0.05) were used. All statistical analyses were performed with Statistica 13.3 (TIBCO Software Inc., Palo Alto, CA, USA).
6
Quantitative Real-Time PCR Analysis
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All data shown in the figures are means ± SE (standard error) of at least 3 independent experiments. The LightCycler software 4.1 (Roche, Basel, Switzerland) was used to analyze real-time PCR data. The results were statistically analyzed using the Student’s t-test. Significance was evaluated at p < 0.1 (*), p < 0.05 (**), and p < 0.001 (***). Asterisks indicate a significant difference between treatments.
7
Quantitative Analysis of VHA Genes in Plants
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Total RNA was isolated using EXTRAzol (BLIRT, Gdańsk, Poland), according to the manufacturer’s instructions. After RNase-free DNase I (Thermo Fischer Scientific, Waltham, MA, USA) digestion, RNA samples were used to cDNA synthesis (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA, USA). The expression of selected VHA genes, encoding V-ATPase subunits, was analyzed using real-time PCR. Reactions were performed in a LightCycler 480 system (Roche, Basel, Switzerland) with a Real-Time 2 × PCR Master Mix SYBR kit (A&A Biotechnology, Gdańsk, Poland). Tonoplast intrinsic protein 41-like (TIP41) and elongation factor 1-alpha (EF1) were chosen as reference genes, according to Migocka and Papierniak [108 (link)].
The qPCR reaction conditions were as follow: 30 s at 95 °C; 40 cycles of: 10 s at 95 °C, 10 s at 58 °C (for CsVHA-A, B, c2, c3, a1, a3), 60 °C (for CsVHA-c1) or 66 °C (for CsVHA-a2), and 12 s at 72 °C; 15 s at 65 °C (for CsVHA-A, B, a1, a3, c2, c3), 68 °C (for CsVHA-c1) or 72 °C (for CsVHA-a2), and 30 s at 40 °C of final cooling. LightCycler software 4.1 (Roche, Basel, Switzerland) was used for data analysis. The sequences of the primers used in the reaction were consistent with those used in our previous publication [7 (link)].
The qPCR reaction conditions were as follow: 30 s at 95 °C; 40 cycles of: 10 s at 95 °C, 10 s at 58 °C (for CsVHA-A, B, c2, c3, a1, a3), 60 °C (for CsVHA-c1) or 66 °C (for CsVHA-a2), and 12 s at 72 °C; 15 s at 65 °C (for CsVHA-A, B, a1, a3, c2, c3), 68 °C (for CsVHA-c1) or 72 °C (for CsVHA-a2), and 30 s at 40 °C of final cooling. LightCycler software 4.1 (Roche, Basel, Switzerland) was used for data analysis. The sequences of the primers used in the reaction were consistent with those used in our previous publication [7 (link)].
8
Real-time PCR for Bordetella pertussis
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Real-time PCR assay was used for detection B. pertussis using the primers and TaqMan probe described by Kosters et al. (18 (link)). PCR was performed using a BHQ quencher probe at 100 μM and 50 μM of primers in a final volume of 20 μL and five microliters of the extracted DNA were combined with 15 μL of the master mix. The thermal cycling program was set at 95°C for 10 seconds, 60 cycles of 5 seconds at 95°C, 5 seconds at 57°C and 30 seconds at 72°C; and final extension. All cycles were performed in Light Cycler® 2.0. Instrument and data was analyzed with the LightCycler® Software 4.1 (Roche Diagnostic, Deutschland-Mannheim, Germany).
9
Multiplex RT-PCR for Respiratory Viruses
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RT-PCR for Influenza-A, Influenza-B, RSV-A, RSV-B and Adenovirus virus were performed using a BHQ quencher probe at 125 nM and 250 nM of primers in a final volume of 20 μl. Five microliters of the extracted RNA was combined with 15 μl of the master mix. RT-PCR conditions was 60 cycles of 15 s at 95 °C and 45 s at 60 °C. This process was performed in Light Cycler® 2.0 Instrument and the data was analyzed with the LightCycler® Software 4.1 (Roche Diagnostic, Deutschland-Mannheim, Germany). The primers and the probe for Influenza A and B were described by Selvaraju et al., 2010 [23 (link)], for RSV-A and RSV-B were described by Liu W. et al., 2016 [24 (link)] and for Adenovirus was described by Heim et al., 2003 [25 (link)].
For he case of Parainfluenza 1 virus, Parainfluenza 2 virus and Parainfluenza 3, the primers and conditions for RT-PCR were described by Coiras et al., 004 [26 (link)].
For he case of Parainfluenza 1 virus, Parainfluenza 2 virus and Parainfluenza 3, the primers and conditions for RT-PCR were described by Coiras et al., 004 [26 (link)].
10
Transcriptomic Analysis Protocol
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For RNA Purification, the NucleoSpin RNA Mini Kit (PN: REF 740955.50) by Machery-Nagel (Düren, Germany) was used. The cDNA was synthesized using Bioscript reverse transcriptase, oligo dT primers, and random hexamer primers (Bioline Lückenwalde, Germany) according to the manual. Gene expression was monitored using the nCounter® FLEX (NCT-SYST-FLEX) system and nSolver® software (Ver. 4.0) by NanoString Technologies Inc. (Seattle, USA) and a LightCycler® 2.0 instrument controlled by LightCycler® Software 4.1 by Roche Molecular Systems (Basel, Switzerland). Measurement with the nCounter® system was performed as previously described [10 (link)] using RNAs from 3 independent experiments each. Pairs were formed with each sample and its corresponding control. Relative expression was calculated from the experimental treatment relative to the control of the same experiment. To quantify relative expression, RPL13 was applied as a housekeeping gene as it was most stable in our earlier experiments [10 (link)]. Data were logarithmized to base two, and this log2Fc value used as a measure for expression changes. Quantitative real-time polymerase chain reaction (qRT-PCR) was done as previously reported using RPL13 as a housekeeping gene [10 (link)]. Relative expression was calculated by the 2∆CT method [30 (link)].
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