Ileal epithelial cells and lamina propria mononuclear cells were isolated from ileal tissue as previously described and stored in RLT buffer (Qiagen). RNA was extracted and purified using RNeasy Plus Mini Kit (Qiagen) and quantified by Nanodrop prior to reverse transcription with iScript cDNA synthesis kit (Bio-Rad). qPCR was performed on an Applied BioSciences Quant Studio 6 Flex Real-time PCR (Applied Biosystems) using PerfeCTa SYBR Green Fast mix, Low ROX (Quanta Biosciences). The thermocycler program was as follows: initial cycle of 95°C for 60 s, followed by 40 PCR cycles at 95°C for 5 s, 60°C for 15 s, 72°C for 15 s. Relative levels of the target genes were determined by calculating the ΔCt to housekeeping gene hprt expression. Primer sequences can be found in Table S3 .20 (link),69 (link)–72 (link)
Perfecta sybr green fastmix low rox
Manufactured by Quanta Biosciences
Sourced in United States
PerfeCTa SYBR Green FastMix Low ROX is a ready-to-use reaction mix for quantitative real-time PCR applications. It contains SYBR Green I dye, hot-start Taq DNA polymerase, and optimized buffer components.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy plus mini kit, by Qiagen (4 mentions)
Quantstudio 6 flex real time pcr, by Thermo Fisher Scientific (3 mentions)
Rlt buffer, by Qiagen (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (2 mentions)
7500 real time pcr, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Ncode vilo mirna cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Express sybr greener supermix, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Qiazol, by Qiagen (1 mentions)
Spss statistics software, by IBM (1 mentions)
Turbo dna free, by Thermo Fisher Scientific (1 mentions)
Stratagene mx3000p cycler, by Agilent Technologies (1 mentions)
30 protocols using perfecta sybr green fastmix low rox
1
Ileal Epithelial and Lamina Propria Cell Isolation
2
Quantifying miRNA and mRNA Expressions
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Total RNA was isolated using QIAzol (Qiagen) and complementary DNA synthesized (for miRNA: NCode VILO miRNA cDNA synthesis kit; for non-miRNA: SuperScript VILO cDNA synthesis kit; both Invitrogen). MiR qPCR was performed using Express SYBR GreenER Supermix with premixed ROX (Invitrogen). SNORD95 was used as housekeeping gene for miR to normalize the Ct values (ΔCt), and relative expression was calculated using the 2−ΔΔCt method. Primers for miRNA qPCR for miR-25, miR-93, miR-106b, and SNORD95 were purchased from Qiagen. For mRNA, QPCR was performed using PerfeCTa SYBR Green fastMix low Rox (Quanta Biosciences). Ct values were normalized to housekeeping genes such as HPRT, GAPDH, and Rps29 (ΔCt), and relative expression was calculated using the 2−ΔΔCt method. The list of primers can be found in Supplementary Table 1 .
3
Transcriptional Analysis of P. aeruginosa Biofilms
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WT PA14 and mutant strains were harvested from planktonic cultures (OD600 = 1.0) or from colony biofilms grown for 72 h. RNA was purified using the Zymo Research kit, and the preparations were subsequently treated with DNAse (TURBO DNA-free; Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and quantified using PerfeCTa SYBR Green FastMix Low ROX (Quanta Biosciences, Beverly, MA, USA).
4
qRT-PCR Analysis of V. vulnificus
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A previously described qRT‐PCR protocol was adapted for all in situ expression studies [Williams et al., 2014a ]. Gene expression of V. vulnificus strain CMCP6 was examined, in triplicate, for each sampling time using PerfeCTa SYBR green FastMix, Low ROX (Quanta Biosciences, Beverly, MA). Normalization of target genes was performed using glyceraldehyde‐3‐phosphate dehydrogenase, an endogenous control, to correct for sampling error. The Pfaffl equation was used to calculate fold change in gene expression, accounting for differences in the primer pairs, utilizing a previously described PCR efficiency analysis [Pfaffl, 2001 ]. Gene expression results were analyzed comparing each sample time to the previous sample time, where t = 0 was immediately upon chamber deployment; for example, x = 12 represents fold change of 12 h to 0 h and x = 24 represents fold change of 24 h relative to 12 h. Nonparametric Mann‐Whitney rank‐sum tests were used to determine significance between target transcripts with adjusted p values calculated using the Bonferonni method. All gene expression data were analyzed using GraphPad Prism (version 5.0; GraphPad Software Inc.). Pearson's product‐moment correlational analysis was performed on gene expression and environmental parameters in SPSS Statistics software (version 24, IBM Corp., Armonk, NY, USA) and output visualized using GraphPad Prism.
5
Pseudomonas aeruginosa RNA Extraction
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P. aeruginosa strains were harvested from HCD planktonic cultures (OD600 = 2.0) and from mature (5-day old) colony biofilms. RNA was purified using Trizol (Ambion), and subsequently, DNAse treated (TURBO DNA-free, Thermo Fisher). cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen) and quantified using PerfeCTa SYBR Green FastMix Low ROX (Quanta BioSciences).
6
Quantitative Gene Expression Analysis
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Gene expression of selected genes was determined using two biological replicas by real-time PCR on a Stratagene Mx3000P cycler (Agilent Technologies). The qPCR reaction (10 μL) consisted of gene-specific primers (Supplementary Table 10 ), PerfeCTa® SYBR® Green FastMix Low ROX™ (Quanta Biosciences), and the template. The cycling conditions were 95 °C/3 minutes followed by 95 °C/15 seconds, 60 °C/30 seconds, and 72 °C/30 seconds for 40 cycles. Primers were designed using PRIMER3 (www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/ ). Data were analyzed using the 2−∆∆Ct method37 (link) and presented as relative levels of gene expression, using ubiquitin (Solyc07g064130) and EF1α (Solyc06 g005060) genes as internal standards. Expression values are the mean of two biological replicates.
7
Quantifying Bacterial DNA in Air Samples
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qPCR on extracted DNA from area air samples was performed in a QuantStudio 7 Flex Real-Time PCR System using MicroAmp Fast Optical 96-well and 384-well reaction plates (0.1ml), MicroAmp optical adhesive film, (all Applied Biosystems) and PerfeCTa SYBR Green FastMix, Low Rox (Quanta Biosciences). Each reaction contained 10ul of 2X Master Mix, 4 ul of DNA template, 625 nM of each primer (IDT), and PCR grade water to a final volume of 20 ul. Amplification was comprised of a 10-minute activation step at 95°C, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and a fluorescence measurement. Melting curve analysis was done by monitoring fluorescence throughout incremental increases of temperature from 60°C to 95°C.
The qPCR primers (1369F-1492R) [22 (link)] target regions flanking V9 of the 16S rRNA gene. The standard curve was made using a serially diluted plasmid that contains nt 1369 to 1492 of an E. coli 16S rRNA gene. The concentrations of unknowns are calculated from CT values using the equation generated from plotting the standard curve. All samples are run in triplicate, including the standard curve, a set of non-template controls (NTC), and inhibitor controls (known positives + unknown DNA).
The qPCR primers (1369F-1492R) [22 (link)] target regions flanking V9 of the 16S rRNA gene. The standard curve was made using a serially diluted plasmid that contains nt 1369 to 1492 of an E. coli 16S rRNA gene. The concentrations of unknowns are calculated from CT values using the equation generated from plotting the standard curve. All samples are run in triplicate, including the standard curve, a set of non-template controls (NTC), and inhibitor controls (known positives + unknown DNA).
8
Ileal Epithelial and Lamina Propria Cell Isolation
9
RNA Quantification via qPCR
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RNA was extracted from cells grown in six‐well plates using Nucleospin II RNA kit (Macherey‐Nagel, Dueren, Germany; www.mn-net.com ). DNase treatment was performed on‐column during RNA extraction. One microgram of RNA was reverse transcribed with the qScript cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD; www.quantabio.com ). PCR was performed on a BioRad iCycler or an AB 7500 using PerfeCTa SYBR Green FastMix for iQ or PerfeCTa SYBR Green FastMix Low Rox (both Quanta Biosciences), respectively. Primer sequences are shown in Supporting Information Table S1. Relative quantitation was performed using the method by Pfaffl 24 and β‐actin as a reference gene from technical duplicates or triplicates of at least three independent experiments.
10
Quantitative RT-PCR Analysis of Colonic Tissue
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RNA from colonic tissue was extracted using RNeasy kits (Qiagen, Copenhagen, Denmark), and cDNA synthesis (QuantiTect Reverse Transcription Kit, Qiagen) was performed according to manufacturer’s protocols, as previously described [16 (link)]. Primers are listed in Table 1 . PerfeCTa SYBR Green FastMIX Low ROX (Quanta Bioscience, Gaithersburg, MD, USA) was used for qPCR using the following program: initial denaturation step at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 20 s. Relative expression was calculated using the ΔΔCT method using Gapdh as a reference gene.
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