RNA was obtained from Sertoli cells using the method of Chomczynski and Sacchi [40 (link)]. Complementary DNAs were synthesized from Sertoli cell RNA by reverse transcription as previously described [41 (link)]. For quantitative PCR (qPCR), oligonucleotide primers (Table 3 ) were designed using Primer Express v. 1.5 (ABI Prism; Applied Biosystems, Foster City, CA). Primer sequences were compared by a BLAST search to ensure there was no homology with other rat genes and were independently validated [42 ]. The qPCR amplifications were performed in the ABI Prism 7900HT Sequence Detection System v 2.3 (Applied Biosystems) as previously described [41 (link)]. Ppia (peptidylprolyl isomerase A, commonly known as cyclophilin) or Gapdh was used as an endogenous control. The means (±SEM) of three to five individual experiments were determined for each treatment group for each gene of interest.
Primer express v1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Primer Express v1.5 is a software application designed for the design of primers and probes for real-time PCR experiments. The software automates the process of primer and probe selection, providing users with a set of optimized oligonucleotide sequences that meet specified criteria, such as melting temperature, GC content, and secondary structure formation.
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Lab products found in correlation
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Abi 7500 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Abi sequence detection 1, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Ribopure bacterial rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Random primer, by Promega (1 mentions)
Improm 2 reverse transcriptase, by Promega (1 mentions)
Experion automated electrophoresis station, by Bio-Rad (1 mentions)
Dna free kit, by Thermo Fisher Scientific (1 mentions)
E z n a total rna kit 1, by Omega Bio-Tek (1 mentions)
Superscript 3 kit, by Thermo Fisher Scientific (1 mentions)
Primer blast, by Thermo Fisher Scientific (1 mentions)
Total rna isolation kit, by Omega Bio-Tek (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Primescript cdna synthesis kit, by Takara Bio (1 mentions)
11 protocols using primer express v1
1
Quantitative PCR Analysis of Sertoli Cell Gene Expression
2
Quantitative RT-PCR for Gene Expression
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The primers sdiARTF and sdiARTR used for the real-time PCR assays were designed by using Primer Express v1.5 (Applied Biosystems) (see Table S1 in the supplemental material). Primer validation and reaction mixture preparation were done as previously described (30 (link)). Quantitative real-time reverse transcriptase PCR (qRT-PCR) was performed in a one-step reaction using the ABI 7500 sequence detection system (Applied Biosystems). Using the ABI sequence detection 1.2 software (Applied Biosystems), data were collected and normalized to endogenous levels of rpoA. Data were analyzed by using the comparative critical threshold cycle (CT) method and are presented as fold changes compared to levels of the WT strain grown to the early log growth phase without AHLs. Error bars represent the standard deviations of the CT values.
3
Construction of 3x FLAG-tagged SdiA EHEC Strain
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The methods for PCR amplification, plasmid purification, and transformations were performed using standard protocols as previously described (27 ). Oligonucleotide primers (see Table S1 in the supplemental material) were designed by using Primer Express v1.5 (Applied Biosystems). The wild-type EHEC strain expressing chromosomally 3× FLAG-tagged SdiA was constructed using recombinant DNA techniques as described previously in references 28 (link) and 29 (link). Briefly, PCR product was amplified using Phusion high-fidelity DNA polymerase (Thermo Scientific), sdiAFLAGF and sdiAFLAGR primers, and pSUB11 (Knr) plasmid as the template. PCR product was digested with DpnI to remove the template DNA and then gel purified (Qiagen). Cells of the wild-type EHEC strain transformed with the helper plasmid pKD46 were prepared for electroporation and transformed with the resulting gel-purified PCR products. Colonies were screened for ampicillin sensitivity and kanamycin resistance. Successful recombinational transfer of the FLAG sequence into the chromosomal sdiA gene of positive colonies was confirmed by PCR amplification of the integrated region using primers sdiAUP and sdiADOWN. The kanamycin cassette was removed with the resolvase plasmid pCP20. PCR amplification and DNA sequencing were performed for final verification of the resolved chromosomal 3× FLAG-tagged SdiA EHEC strain, YNN04.
4
Quantification of gene expression in treated HPMCs
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Total ribonucleic acid (RNA) was extracted from treated HPMCs using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The total RNA (1 µg) was reverse transcribed to complementary deoxyribonucleic acid (cDNA) using a PrimeScript cDNA Synthesis Kit (TaKaRa Shuzo Co. Ltd., Otsu, Japan). All primers for the quantitative reverse transcription PCR (qRT-PCR) (Suppl. Table 1 ) were designed using Primer Express v.1.5 (Applied Biosystems, Foster City, CA, USA) software. The qRT-PCR was performed, in duplicate, on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems). All samples were analyzed using the comparative Ct method (2−ΔΔCt) for the relative quantification of gene expression and normalization with respect to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression.
5
Quantitative Analysis of Virulence Gene Expression
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Overnight cultures of WT (in the absence or presence of 50 µM epinephrine or norepinephrine) and ΔqseC, ΔqseE and ΔqseEC strains were diluted 1:100 and grown aerobically at 37°C in low-glucose Dulbecco’s modified Eagle medium (DMEM) (Gibco) to the exponential-growth phase (optical density at 600 nm [OD600] = 0.7). RNA was extracted from three biological samples using a RiboPure bacterial RNA isolation kit (Ambion) following the manufacturer’s guidelines. The primers used in the real-time assays were designed using Primer Express v1.5 (Applied Biosystems) (63 (link)) and were validated for amplification efficiency and template specificity. Quantitative real-time PCR (qRT-PCR) was performed as previously described (21 (link)) in a one-step reaction using an ABI 7500 sequence detection system (Applied Biosystems). Data were collected using ABI Sequence detection 1.2 software (Applied Biosystems).
All data were normalized to an rpoA (RNA polymerase subunit A) endogenous control and analyzed using the comparative cycle threshold (CT) method. Virulence gene expression was presented as fold changes over the WT expression level. Error bars indicate the standard deviations of the fold change values. The Student unpaired t test was used to determine statistical significance.
All data were normalized to an rpoA (RNA polymerase subunit A) endogenous control and analyzed using the comparative cycle threshold (CT) method. Virulence gene expression was presented as fold changes over the WT expression level. Error bars indicate the standard deviations of the fold change values. The Student unpaired t test was used to determine statistical significance.
6
Quantitative Gene Expression Analysis
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The total RNA was extracted from Trizol (Invitrogen, Carlsbad, CA, USA)-treated pericytes in accordance with the manufacturer’s instructions. One microgram of total RNA was transcribed reversely by using the PrimeScript cDNA Synthesis kit (Takara Shuzo Co., Otsu, Japan). All primers for realtime PCR (Supplemental Table S1 ) were designed using the Primer Express V1.5 software (Applied Biosystems, Foster City, CA, USA). Quantitative realtime PCR was performed on the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). All of the samples were calculated using the comparative Ct method for the relative quantification of gene expression and normalization with regard to the GAPDH expression.
7
Profiling Gene Expression in Thymus
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The expression of c-Myc, TCF3, TP63, KRT5, BAX and BCL2 in the thoracic and cervical thymus was investigated by quantitative PCR (qPCR). For this purpose, fifty milligrams of thymus were disrupted using a TissueLyser II (Qiagen, Hilden, Germany) with stainless steel beads in 1 mL of TRIzol (Invitrogen, Thermo Fisher Scientific). Total RNA was purified from any residual genomic DNA with a DNA-free kit (Ambion). The integrity of the RNA was confirmed by the Experion Automated Electrophoresis Station (Bio-Rad, Hercules, CA), and the concentration was measured by a spectrophotometry. cDNA was synthesised from 1 µg of total RNA using ImProm-II reverse transcriptase (Promega, Madison, WI) and random primers (Promega). To determine the amount of the specific target genes, cDNA was subjected to qPCR using the SYBRGreen method and the IQ5 (Bio-Rad) detection system. The primer sequences were designed using Primer Express v 1.5 (Applied Biosystems, Thermo Fisher Scientific) (Table 1). The peptidylprolyl isomerase A (cyclophilin A, PPIA) gene was used as a housekeeping control gene, as previously reported [14] . The expression level of each target gene was calculated using the 2 -ΔCq method, where ΔCq = Cqtarget gene -Cqhousekeeping gene [15] .
8
Quantifying mRNA Expression in hOPCs
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Total RNA was isolated from hOPCs following mitogen withdrawal and real-time RT-PCR performed as described in ref. 18 (link). Briefly, mRNA was isolated using an E.Z.N.A Total RNA Kit I (Omega Bio-Tek, Norcross, GA) according to the manufacturer’s protocols and complementary DNA prepared (SuperScript III Kit; Invitrogen, Carlsbad, CA). Human-specific primers for SYBR green-based PCR were designed using Primer Express (v1, Applied Biosystems, Foster City, CA). Primer sequences are shown in Supplementary Table 2 . Gene expression was calculated by normalizing to glyceraldehyde 3-phosphate dehydrogenase and performing ΔΔCt analysis. Statistical significance was tested on log 2-transformed data using repeated-measures one-way ANOVA followed by Tukey’s post-test.
9
RNA Extraction and qRT-PCR Analysis
10
Quantitative RNA Expression Analysis in Kidney Tissues
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Total RNA was extracted from kidney tissues using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed to cDNA using the PrimeScript cDNA synthesis kit (TaKaRa, Otsu, Japan). qRT-PCR was performed on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the SYBR Green PCR master mix (Life Technologies). The results were analyzed using the comparative Ct method for relative quantification of gene expression. All primers used in the qRT-PCR were designed using Primer Express V1.5 software (Applied Biosystems) and were as follows: Neutrophil gelatinase-associated lipocalin (NGAL) (forward 5′-GCC ACT CCA TCT TTC CTG TTG-3′ and reverse 5′-GGG AGT GCT GGC CAA ATA AG -3′); IL-1β (forward 5′-TCG TGC TGT CGG ACC CAT AT-3′ and reverse 5′-GGT TCTC CTT GTA CAA AGC TCA TG-3′); IL-6 (forward 5′-CCC ACC AAG AAC GAT AGT CAA TT-3′ and reverse 5′-CAC CAG CAT CAG TCC CAA GA-3′); transforming growth factor (TGF)-β(forward 5′-GGC TGT GGC CAT CAA GAA TT-3′ and reverse 5′-GCA GAG GGA AGA GTC AAA CAT GT-3′); TNF-α (forward 5′-GAC TAG CCA GGA GGG AGA ACA G-3′ and reverse 5′-CAG TGA GTG AAA GGG ACA GAA CCT-3′); and β-actin (forward 5′-ACC ACC ATG TAC CCA GGC ATT-3′ and reverse 5′-CCA CAC AGA GTA CTT GCG CTC A-3′).
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