RNA from bacterial cultures grown to the mid-logarithmic phase of growth was extracted using TRIzol reagent (Invitrogen), treated with DNase I (Invitrogen) and transcribed into cDNA using the SuperScript III Reverse Transcriptase (Invitrogen). Quantitative real-time PCR (qPCR) was performed with the SensiFAST SYBR No-ROX Kit (FroggaBio) using the CFX96 Real-Time System (Biorad). Thermocycling parameters were as follows: 95 °C for 2 min hot start, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min followed by a melting curve. Primers to genes of interest were designed using PrimerQuest software from Integrated DNA Technologies (IDT). Primers were tested for performance in qPCR with a cDNA concentration gradient, and those with slopes between −3.3 and −3.7, the efficiency of ∼1.0 and R2 of ∼1.0 were used in the qPCR studies. All primers employed here are listed in Table 2 . Expression of tsiV1 relative to 16 s rRNA control was determined by the 2−ΔΔCT method using the CFX Manager Software (Biorad). The resulting relative quantification (RQ) values of all samples were normalised against expression of strain C6706.
Primerquest software
Manufactured by Integrated DNA Technologies
Sourced in United States, Australia
PrimerQuest software is a tool developed by Integrated DNA Technologies for designing primers and probes for PCR and other nucleic acid amplification techniques. The software uses advanced algorithms to analyze DNA sequences and generate optimal primer and probe sequences based on user-specified parameters.
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Lab products found in correlation
Emeraldamp gt pcr master mix, by Takara Bio (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Gradient thermal cycler, by Eppendorf (3 mentions)
Sensifast sybr no rox kit, by FroggaBio (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Cfx manager software, by Bio-Rad (2 mentions)
Superscript 3 cdna synthesis kit, by Qiagen (2 mentions)
Sybr green pcr master mix, by Bio-Rad (2 mentions)
Myiq single color real time pcr detection system, by Bio-Rad (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Qiaamp rna blood mini kit, by Qiagen (2 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
40 protocols using primerquest software
1
Quantitative gene expression analysis
2
Detailed Cultivation Protocols for Xylella fastidiosa
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Bacterial strains, plasmids, primers, and culture conditions.
Bacterial strains and plasmids used in this study are listed in Table 1. All primers (Table 2) were designed by PrimerQuest software (Integrated DNA Technologies). X. fastidiosa type strain Temecula and a more aggressive strain, WM1-1 (Oliver et al. 2014; Parker et al. 2012) , were used as the WT strains. All X. fastidiosa strains and mutants were cultured on PW or PD3 plates (Davis et al. 1981) or PD2 broth (Davis et al. 1980 ) at 28°C. All cell suspensions of X. fastidiosa used in this study were prepared as follows. The 7-day-old bacterial cultures were scraped from PW agar medium plates, were suspended in PD2 broth, and were diluted to an optical density at 600 nm (OD 600 ) of 1.0. All E. coli strains were cultured on Luria-Bertani (LB) (Difco, BD Company) plates or in LB broth. To select transformants, 100 µg of ampicillin and 50 µg of kanamycin per liter were used.
Bacterial strains and plasmids used in this study are listed in Table 1. All primers (Table 2) were designed by PrimerQuest software (Integrated DNA Technologies). X. fastidiosa type strain Temecula and a more aggressive strain, WM1-1 (Oliver et al. 2014; Parker et al. 2012) , were used as the WT strains. All X. fastidiosa strains and mutants were cultured on PW or PD3 plates (Davis et al. 1981) or PD2 broth (Davis et al. 1980 ) at 28°C. All cell suspensions of X. fastidiosa used in this study were prepared as follows. The 7-day-old bacterial cultures were scraped from PW agar medium plates, were suspended in PD2 broth, and were diluted to an optical density at 600 nm (OD 600 ) of 1.0. All E. coli strains were cultured on Luria-Bertani (LB) (Difco, BD Company) plates or in LB broth. To select transformants, 100 µg of ampicillin and 50 µg of kanamycin per liter were used.
3
Pig-specific Primer Design for Metabolic Genes
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The primers were purchased from Sigma-Aldrich and Integrated DNA Technologies, and were designed using both PrimerQuest software (Integrated DNA Technologies, Carlsbad, CA) and Primer-BLAST (NCBI). Primer oligonucleotides for ACO, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hepatic CPTIα, CPTIβ, CPTII, mHMGCS, PPARα, ACCα, ACCβ, and MCD were designed with the pig-specific primers except CPTII (human; Supplementary Table 1 ). Primer pairs were selected for optimum annealing temperatures and negligible secondary structure.
4
RNA Extraction and cDNA Synthesis
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RNA was extracted from samples using the RNeasy Mini Kit (Qiagen, Germantown, MD). Samples were then treated with RNase-free DNase I and reversely transcribed using oligo-dT (SuperScript III cDNA synthesis kit from Qiagen, cat# 74104). Primers (Additional file 1 : Table S1) used were specifically designed with PrimerQuest software (Integrated DNA Technologies).
5
Gene Expression Quantification by RT-PCR
6
Copper Responsive Gene Expression
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Gene expression of copA and copB was analyzed under 200, 250, and 350 µM CuSO 4 ) and Cudepleted (150 µM bathocuproine sulfate [BCS]) conditions. X. fastidiosa WT cells were grown in 5 ml of PD2 media with initial OD 600 = 0.01. After 7 days, bacteria were collected by centrifugation at 13,000 × g for 3 min. Total RNA from each treatment was extracted using the Quick-RNA MiniPrep kit (Zymo Research, Irvine, CA). Contaminant DNA in samples was removed by treatment with DNase I (Thermo Scientific, Pittsburgh, PA). One microgram of RNA of each sample was used to synthesize single-strand cDNA by reverse transcription using the Maxima First Strand cDNA synthesis kit for reverse transcription-quantitative PCR (RT-qPCR) (Thermo Scientific, Pittsburgh, PA). cDNA samples were diluted five times with RNA/DNA-free water. Primers used for copA and copB were designed using PrimerQuest software (Integrated DNA Technologies, Coralville, IA). gyrB was used as an internal control, as previously described (Cruz et al. 2014) (link). The qPCR was carried out with SYBR green (Bio-Rad, Hercules, CA) using ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA). The fold change in gene expression compared with WT was calculated according to the 2 -ΔΔCT method (Livak and Schmittgen 2001).
7
Quantifying gene expression in retina
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mRNA levels of selected genes were measured using real-time PCR with cDNA extracted from retinas as templates. Primers (Table 2 ) were designed using Primer Quest software (Integrated DNA Technologies, Coralville, IA, USA) spanning the intron–exon boundary to amplify the corresponding mRNAs without amplifying potentially contaminating genomic DNA. Real-time PCR was carried out with the SYBR green PCR master mix (Bio-Rad Lab., Hercules, CA, USA) using the MyiQ Single-Color Real-Time PCR detection system (Bio-Rad Lab., Hercules, CA, USA) according to the manufacturer’s instructions. Electrophoresis of PCR products was performed to identify that a single band of the correct size had been amplified.
8
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted using the TRizol reagent (Invitrogen) according to the manufacturer's instructions. RNA concentrations were determined using a NanoDrop spectrophotometer (Thermo Scientific). 1μg of RNA from each sample was treated with DNase I (Invitrogen), and transcribed into cDNA using the SuperScript III Reverse Transcriptase (Invitrogen). Quantitative real-time PCR (qPCR) was performed with SensiFAST SYBR No-ROX Kit (FroggaBio), using the CFX96 Real-Time System (Biorad). Thermocycling parameters were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, followed by a melting curve. Primers against the different genes of interest were designed using the PrimerQuest software from Integrated DNA Technologies (IDT). Primers were tested for performance in qPCR with a cDNA concentration gradient, and those with slopes between −3.3 and −3.7, efficiency of ∼1.0, and R2 of ∼1.0 were used in the qPCR studies (primer sequences used in the study are summarized in Table 1 ). The expression levels of the different targets in relation to the endogenous 16S rRNA gene control was determined by the 2−ΔΔCT method using the CFX Manager Software (Biorad). The relative quantification (RQ) values of all samples were normalized against the expression of the 16S control for each target.
9
Gene Expression Analysis via qPCR
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Total RNA was extracted with the RNeasy kit (Qiagen, Germantown, MD, USA), treated with RNase-free DNase I (Qiagen), and reverse transcribed with oligo-dT using the SuperScript III cDNA synthesis kit (Invitrogen). Quantitative PCR was performed with the QuantiTect SYBR Green PCR Kit (Qiagen). Specific primers (Supplementary Table 1 ) for each gene were designed with the PrimerQuest software (Integrated DNA Technologies, Coralville, IA, USA), and the cDNA sequences of each gene (GenBank, NCBI, NIH) were used to produce 100–250 bp PCR amplicons that span one or more exon/intron boundaries.
10
Canine Immune Gene Expression Analysis
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Total RNA was extracted using the RNeasy Mini RNA Isolation kit (Qiagen) according to manufacturer’s instructions. Contaminating genomic DNA was removed using the RNase-Free DNAse Digestion set (Qiagen). Total eluted RNA was reverse transcribed into cDNA using Superscript III Reverse Transcriptase and Random Primers (both from Life Technologies, Inc.). Real-time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems). The primer sequences for canine GAPDH, canine granzyme B, and canine perforin have been previously published (43 (link), 44 (link)). Forward and reverse primers were designed for canine granulysin (Accession XM_845424.3) using PrimerQuest software from Integrated DNA Technologies: canine granulysin (forward): 5′-TGTGTAGTGTTGCCCAGTTT-3′; canine granulysin (reverse): 5′-CTCCTTGGACACCTACTTGATG-3′. The reactions were performed on an Agilent MX3000P Real-Time PCR machine (Agilent) with the following cycling conditions: 2 min at 50°, 10 min at 95°, followed by 40 cycles of 15 s at 95° and 1 min at 60°, and a dissociation (melting) curve (15 s at 95°, 1 min at 60°). Relative gene expression was determined using the 2−ΔΔCt method, with GAPDH as the reference housekeeping gene.
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