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7500 sequence detector

Manufactured by Thermo Fisher Scientific
Sourced in United States

The 7500 Sequence Detector is a real-time PCR instrument designed for gene expression analysis, SNP genotyping, and other genetic applications. It features a 96-well sample block and uses fluorescence detection to monitor the amplification of DNA samples during the PCR process.

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45 protocols using 7500 sequence detector

1

Quantification of Lactobacillus in Fecal Samples

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E.Z.N.A Stool DNA KIT (Omega Bio-tek, USA) was used to extract Total fecal DNA. qPCR on a 7500 Sequence Detector (Applied Biosystems, CA, USA) was used to calculate the number of Lactobacillus sp. Relative gene copy number of Lactobacillus in genomic DNA extracted from fecal samples were calculated by the qPCR on a 7500 Sequence Detector (Applied Biosystems, CA, USA) and compared with 16s rRNA gene.Samples were quantified in 20 μl reactions using ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Vazyme, Shanghai, China). Lac-F (5’-TGGAAACAGGTGCTAATACCG-3’) Lac-R (5’- CCATTGTGGAAGATTCCC-3’) 16S rRNA-F (5’-ACTCCTACGGGAGGCAGCAGT-3’) 16S rRNA-R(5’-GTATTACCGCGGCTGCTGGCAC-3’). All measurements were performed in duplicate.
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2

Quantitative Gene Expression Analysis

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Total RNA was extracted from PECs with Trizol (Invitrogen, Eugene, Oregon, USA). RNA extraction from human and murine synovial tissues was performed using RNA-Bee (Amsbio, Milton, Abingdon, U.K.). Isolated RNA was reverse transcribed using Super Script II reverse transcriptase (Invitrogen, Eugene, Oregon, USA). The gene specific Taqman assay (Applied Biosystems, Waltham, MA, USA) was used for amplification and detection of different genes with Applied Biosystems 7500 sequence detector. Gene expression fold changes of different mRNA of treated samples were quantified relative to the untreated after normalizing to 18s RNA, as previously described10 (link).
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3

Quantitative gene expression analysis

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Total RNA was isolated using Tri-Reagent (Life Technologies). RNAs were quantified and cDNAs were synthesized using ImProm-II™ Reverse Transcription System (Promega). TaqMan gene expression assays were purchased from Applied Biosystems. GAPDH was used as a housekeeping gene. cDNAs were subjected to real time qPCR using HotStart-IT or VeriQuest Probe qPCR Master Mixes (Affymetrix) and TaqMan technology (7500 Sequence Detector, Applied Biosystems). The relative mRNA levels were quantified using Applied Biosystems software.
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4

RNA Isolation and Real-Time PCR Analysis

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Cells were grown as described above in 1.5 mL TSB, harvested and broken with a Mini-Beadbeater (Biospec Products) at maximum speed for 30 s. After incubation on ice for 5 min, the samples were centrifuged; and the supernatant was used to isolate total RNA according to the manufacturer’s instructions (Qiagen). After DNase treatment with a TURBO DNA-free TM kit (Ambion), approximately 2 μg of total RNA were reverse-transcribed with a PrimeScript RT reagent kit (Qiagen). The cDNA was used as a template for real-time PCR using SYBR-green PCR reagents (Roche). Reactions were performed in a MicroAmp Optical 96-well reaction plate using a 7500 Sequence Detector (Applied Biosystems). Primers used are listed in Supplementary Table 8. All RT-PCR experiments were performed in triplicate with gyrB as an internal control. All experiments were repeated independently at least three times with similar results.
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5

Gene Expression Analysis of Bacterial Cultures

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Bacteria
were cultured in TSB overnight. The overnight cultures were diluted
(1:100) in fresh TSB or TSB with 10% spent culture of S. epidermidis and grown for 8 h at 37 °C with
shaking. The pellets were collected by centrifuge at 4000g for 10 min and broken by FastPrep-24 (MP 116005500). After centrifugation,
the supernatant was used to isolate the total RNA and further synthesize
complementary DNA according to the manufacturer’s instructions
(Qiagen). The cDNA was used as a template for real-time PCR using
SYBR-green PCR reagents (Roche) with primers listed in Table S1. Reactions were performed in a MicroAmp
Optical 96-well reaction plate using a 7500 Sequence Detector (Applied
Biosystems). All qRT-PCR experiments were performed in triplicate
using gyrB as an internal control gene at the mRNA
level.
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6

Quantitative Real-Time PCR for Gene Expression

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Total RNA was isolated from cells by using TRI Reagent (Sigma, Milan, Italy). The amount and purity of RNA were determined spectrophotometrically. cDNAwas obtained by incubating 2 μg of total RNA with 4 U/μL of M-MLV reverse transcriptase (Promega, San Luis Obispo, CA, USA) according to the manufacturer’s instructions. Quantitative real time PCR (qPCR) was performed as reported in [51 (link)] using the GoTaq® Probe Systems (Promega). The qPCR analysis was carried out in triplicate using an Applied Biosystems 7500 Sequence Detector with the default PCR setting: 40 cycles at 95 °C for 15 s, 60 °C for 60 s. mRNA was quantified with the DDCt method as described [52 (link)]. mRNA levels were normalized to β2 microglobulin as an endogenous control. The primer sequences used are listed in Table 1.
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7

Extraction and Quantification of S. epidermidis RNA

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The S. epidermidis strains were grown to stationary phase. The cells were centrifuged and the pellets were lysed with a FastPrep-96 (MP Biomedicals Products) at 800 rpm for 300 s three times. Supernatants were collected for total RNA isolation according to the manufacturer’s instructions (Qiagen). After DNase treatment with a TRUBO DNA-free kit (Ambion), 1 μg of total RNA was reverse transcribed with a Prime Script RT reagent kit (Qiagen). The cDNA was used as a template for RT-PCR with SYBR Green PCR reagents (Roche). Reactions were performed in a MicroAmp optical 96-well reaction plate with a 7500 sequence detector (Applied Biosystems).
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8

Quantitative Real-Time PCR for Gene Expression

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First-strand cDNA was generated using the SuperScript III First-Strand Synthesis System for RT-PCR Kit (Invitrogen), with random hexamers, according to the manufacturer’s protocol. PCR was performed in a total volume of 25 µl containing 1× Taqman Universal PCR Master mix, no AmpErase UNG, and 2.5 µl of cDNA; pre-designed, gene-specific primers, and probe sets for each gene were obtained from Assay-on-Demande Gene Expression Products (Applied Biosystems).
Real-time PCR reactions were carried out in a two-tube system and in singleplex. The real-time amplifications included 10 min at 95°C (AmpliTaq Gold activation), followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min. Thermocycling and signal detection were performed with 7500 Sequence Detector (Applied Biosystems). Signals were detected according to the manufacturer’s instructions and the relative expression levels were calculated as it has been previously described (9 (link)).
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9

Multiplex qMSP Analysis of Gene Promoter Methylation

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The nine genes (APC, FOXA1, MGMT, RARβ2, RASSF1A, SCGB3A1, SEPT9, SHOX2 and SOX17) promoter methylation levels were assessed by multiplex qMSP, using amplified DNA as template [25 (link),55 (link)]. Primers and probes specifically designed for the modified gene sequence plus the fluorochromes and quenchers used for each probe are listed in Supplementary Table S6. β-Actin was used as reference gene to normalize the DNA quantity of each sample [18 (link)]. 6 µL of WGA amplified DNA and Xpert Fast Probe (GRiSP, Porto, Portugal) were used in each multiplex qMSP reaction. Multiplex qMSP assays were carried out in 96-well plates in triplicate using a 7500 Sequence Detector (Applied Biosystems, Perkin Elmer, CA, USA). Sterile distilled water subjected to WGA was used a negative control and included in all plates. WGA amplified CpGenome™ Universal Methylated DNA (Merck Millipore, Burlington, MA, USA) subjected to six serial dilutions (5× factor dilution) was used to generate a standard curve in each plate, allowing for relative quantification and PCR efficiency evaluation. Efficiency values above 90% were considered. Relative methylation levels were determined as the ratio between the mean methylation levels of each gene and the respective value for β-Actin (the housekeeping gene), multiplied by 1000 for easier tabulation.
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10

Biofilm Formation Genes in S. aureus

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The expression of adhesion and biofilm formation genes in ST188 was detected by RT-PCR. We chose ST398 as the reference group because ST398 is an important host-species-adaptable S. aureus lineage with high virulence but low biofilm formation ability18 (link),21 (link). Complementary DNA was synthesized from total RNA using the QuantiTect reverse transcription system (Qiagen) according to the manufacturer’s instructions. Complementary DNA samples were amplified using the QuantiTect SYBR green PCR kit (Qiagen). Reactions were performed using a 7500 Sequence Detector (Applied Biosystems). We used purified chromosomal DNA at concentrations of 0.005–50 ng/ml to form a standard curve. All quantitative reverse-transcription polymerase chain reaction (qRT-PCR) experiments were performed in duplicate with gyrB as a control.
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