The quantitative PCR method can obtain expression profiles of genes of interest in a high-throughput and accurate manner22 (link). In this work, the same experimental conditions were used44 . RNA isolation was based on the suggested protocol in the TRI Reagent-RNA Kit (Molecular Research Center, Cincinnati, OH, USA). RNA samples for quantitative PCR were pre-treated with DNase I (Promega, Madison, WI, USA). The DNA primers used in the quantitative PCR were designed by Primer Express software (Applied Biosystems, Foster City, CA, USA) and their complements (Table S5 ). DNA was synthesised using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). The quantitative PCR experiments were conducted in an ABI PRISM® 7000 instrument (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix Ex Taq reagent (Takara, Tokyo, Japan). All quantitative PCR experiments were performed with two replicates.
Abi prism 7000 instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI PRISM® 7000 Sequence Detection System is a real-time PCR instrument designed for quantitative gene expression analysis. It features a 96-well thermal cycler and a 4-color optical detection system. The instrument is capable of performing real-time PCR and reverse transcription PCR (RT-PCR) experiments.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq reagent, by Takara Bio (1 mentions)
Dnase 1, by Promega (1 mentions)
Primer express software, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Turbo dnase kit, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master kit, by Thermo Fisher Scientific (1 mentions)
Fastprep, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Lysing matrix b tube, by MP Biomedicals (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Innuprep rna mini kit, by Analytik Jena (1 mentions)
M mulv reverse transcriptase, by New England Biolabs (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Ez1 rna universal tissue kit, by Qiagen (1 mentions)
Improm iitm reverse transcription system, by Promega (1 mentions)
5 protocols using abi prism 7000 instrument
1
Quantitative PCR Gene Expression Profiling
2
RNA Isolation and qRT-PCR Analysis of MRSA
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Total RNA was isolated from study MRSA strains using the method as described previously (60 (link), 61 (link)). Briefly, pelleted MRSA cells (~109 CFU) were resuspended in RLT buffer from RNeasy kit (Qiagen, Germantown, MD, USA), then transferred to lysing matrix B tubes (MP Biomedicals, Irvine, CA, USA), and disrupted using FastPrep (Thermo Fisher, Waltham, WA, USA). After centrifugation at 13,000 rpm at 4°C for 10 min, the supernatant was used for RNA isolation according to the manufacturer’s instructions for the RNeasy kit and then treated with TURBO DNase kit (Thermo Fisher) to remove remaining DNA. DNase-treated RNA (1 μg) was transcribed into cDNA using the SuperScript III first-strand synthesis kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocols. qRT-PCR was performed using an ABI Prism 7000 instrument (Applied Biosystems, Waltham, MA, USA) and a SYBR green PCR master kit (Applied Biosystems) (8 (link)). Primers used in this study are listed in Table S1. A housekeeping gene, gyrB, was used to normalize the transcript quantification. Relative quantification of interesting gene expression was calculated by the threshold cycle (ΔΔCT) method and then normalized versus the relative expression level in the WT strain (4 (link)).
3
qRT-PCR Analysis of ARNT mRNA
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Gene expression was analysed by quantitative reverse transcription – polymerase chain reaction (qRT-PCR). Therefore total RNA was isolated using the innuPREP RNA Mini Kit (Analytik Jena) as described in the supplier’s protocol. Reverse transcription was performed using oligo-dT primer and M-MuLV Reverse Transcriptase (New England Biolabs) in accordance with the manufacturer’s guidelines. ARNT mRNA expression was measured using TaqMan® Gene Expression Assays (#Hs01121918_m1, Applied Biosystems) and compared to endogenous Beta-2-microglobulin (B2M) mRNA expression levels (#Hs00984230_m1, TaqMan® Gene Expression Assays, Applied Biosystems). Quantitative real-time PCR was carried out on an ABI PRISM® 7000 instrument (Applied Biosystems) applying the protocol for comparative relative quantitation (∆∆Ct method).
4
Lung Gene Expression Analysis
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Gene expression in lung tissue of podoplanin, α‐SMA, fibronectin, and collagen was determined by RT‐PCR as follows. Total RNA was extracted from snap‐frozen lung hemilobes using EZ1 RNA Universal Tissue Kit (Qiagen, Hilden, Germany) following manufacturer's instructions. cDNA was prepared with ImProm‐IITM Reverse Transcription System (Promega, Madison, Wisconsin). Quantitative RT‐PCR was performed using PowerUp Green Master Mix SYBR Green PCR master kit (Life Technologies) on a ABIPRISM 7000 instrument (Applied Biosystems). Data were normalized to the housekeeping gene (β‐actin) and relative gene expression was quantified using the 2−ΔCT method. Primer sequences are shown in Supporting Information Table S1 .
5
Quantitative Analysis of m6A Regulators
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Total RNA was extracted by Trizol and reverse transcribed using PrimeScript RT kit. The primers for Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, Ythdf2, and GADPH listed in Table 1 were synthesized by Shanghai Thermo Fisher Biotechnology Co. RT-PCR was performed using SYBR® PreMix Ex TaqTM II kit according to the manufacturer's protocol. The reaction mix (15 μL) contained 7.5 μL of SYBR®PreMix Ex TaqTM II (2×), 0.5 μL of forward primer, 0.5 μL of reverse primer, 0.05 μL of ROX reference dye (50×), 2 μL of template, and 5.5 μL of double-distilled water (DDH2O). RT-PCR was performed on an ABI Prism® 7000 instrument (Applied Biosystems, California, USA) with the following cycling conditions: pre-denaturation at 95 °C for 30 s, followed by denaturation at 95 °C for 5 s and annealing extension at 60 °C for 30 s for 40 cycles. U6 and GADPH were used as internal standards. The relative expression of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 were calculated by the 2-ΔΔCT method, and all experiments were performed independently three times.
Table 1. Primer sequences for RT-PCR. Table 2. Compounds in CDD with OB larger than 30% and DL larger than 0.18, which combined with the results of UHPLC-Q/TOF-MS.
Table 1. Primer sequences for RT-PCR. Table 2. Compounds in CDD with OB larger than 30% and DL larger than 0.18, which combined with the results of UHPLC-Q/TOF-MS.
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