Trizol was used to extract total RNA from the cells. cDNA was synthesized through reverse transcription (Vazyme, Shanghai, China) using the BIO-RAD instrument and with SYBR Mix (Vazyme, Shanghai, China) to perform qRT-PCR.The information on the sequence is provided in Supplementary Material Table S2 .
Reverse transcription
Manufactured by Vazyme
Sourced in China
Reverse transcription is a laboratory technique used to synthesize complementary DNA (cDNA) from a single-stranded RNA template. The core function of this process is to convert RNA into DNA, which can then be used for various downstream applications, such as gene expression analysis, viral detection, and sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Prism v8, by GraphPad (2 mentions)
Hiper sybr premix estaq, by Mei5 Biotechnology (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Rt kit, by Vazyme (2 mentions)
Sybr mix, by Vazyme (1 mentions)
R323 01, by Vazyme (1 mentions)
Trnzol universal reagent, by Tiangen Biotech (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
Abi 7300 real time pcr machine, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr qpcr master mix kit, by Vazyme (1 mentions)
7 protocols using reverse transcription
1
RNA Extraction and qRT-PCR Analysis
2
Quantitative Analysis of miR-124-5p and MIEN1 mRNA
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Primers was designed with Primers 5.0 software in the light of the human miR-124-5p and MIEN1 mRNA sequences in GeneBank, and produced in Shanghai Sangon Biotech Co., Ltd. The following primer sequences were used: miR-124-5p (5’-CGTGTTCACAGCGGACCTTGAT-3’), U6 (forward primer: 5’-GCTTCGGCAGCACATATACTAAAAT-3’, reverse primer: CGCTTCACGAATTTGCGTGTCAT-3’), MIEN1 (forward primer: 5’-CAGTGCTGTGGAGCAGT-3’, reverse primer: 5’-GACGGCTGTTGGTGATCTTT-3’), GAPDH (forward primer: 5’-gagcgagatccctccaa-3’, reverse primer: 5’-actgtggtca tgagtccttc-3’). Total RNA was isolated by Trizol reagent. The purity and concentration of RNA were detected by ultraviolet spectrophotometry. The first strand of cDNA was synthesized by reverse transcription (Vazyme Biotech Co., Ltd., Nanjing, China). GAPDH was the MIEN1 mRNA internal reference gene and U6 as the miR-124-5p internal reference gene on ABI7300 fluorescence quantitative PCR instrument. The relative expression level of target gene RQ = 2-ΔΔCT.
3
Total RNA Extraction and Quantitative PCR
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Total RNA was extracted from cell culture samples using the TRNzol Universal Reagent (Tiangen, W9712) according to the manufacturer’s instructions. The cDNA was synthesized from total RNA (1 μg) using reverse transcription (Vazyme, R323-01). Primer sequences were as follows in Table 1 . PCR amplification was executed by the SYBR Green PCR master mix (LightCycler 480, 30408), and the PCR-amplified gene products were analyzed.
4
Quantifying Antibiotic Resistance Genes
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Residual genomic DNA was completely removed by wipe Mix, and cDNA was synthesized via reverse transcription (Vazyme Biotech Co., Ltd, Nanjing, China). qPCR was performed on the CFX96 real-time PCR detection system (Bio-Rad Laboratories Co., Ltd, Hercules, CA, USA) using HiPer SYBR Premix EsTaq (Mei5 Biotechnology, Co., Ltd, Beijing, China). The mecA primer set was designed using primer Basic Local Alignment Search Tool (BLAST) for the region spanning 36–335 bp, and the housekeeping gene gryB was used as an internal standard for normalization (Table S1 in the supplemental material). The thermal cycling conditions were initial denaturation at 95°C for 2 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The experiment was repeated three times. Fold changes in mecA expression were illustrated using GraphPad Prism v8.0 (GraphPad Software Inc., San Diego, CA, USA). The mecA gene was amplified using the synthesized cDNA as template via conventional PCR. Products were sequenced using Sanger sequencing and the sequences were comparatively analyzed.
5
RNA Extraction and Real-Time PCR Protocol
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Total RNA was extracted from C18-4 cells with Trizol reagent after various treatments. A reverse transcription (RT) kit (Vazyme, Nanjing, China) was used for the reverse transcription of mRNA into cDNA at 42°C for 2 min with 0.6 μg of total RNA, 4 μl of 4× gDNA wiper mix, and 10 μL of RNase-free water. Then, 4 μL of 5× HiScriptII qRT SuperMixII was added to the reaction mixture. The reaction was continued for 15 min at 50°C and 5 s at 85°C. The mRNA primer sequences for real-time PCR are shown in Supplementary Table S1 . Real-time PCR was performed three times with a CFX Connect Real-Time System (BIORAD, Hercules, United States) according to the manufacturer’s instructions.
6
Quantitative PCR Analysis of EMT Markers
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Total RNA was isolated from cells and tissues using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was generated using a Reverse Transcription (RT) kit (Vazyme Biotech Co., Ltd, Nanjing, China), according to the manufacturer's protocol. qPCR was performed using a SYBR qPCR Master Mix kit (Vazyme Biotech Co., Ltd.) in an ABI 7300 real-time PCR machine (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. qPCRs were performed as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The relative expression of each mRNA of interest were normalized to endogenous control and analyzed using the 2−ΔΔCq method (29 (link),30 (link)). Epithelial (E-)cadherin, β-catenin, fibronectin, vimentin and β-actin primers were as follows: E-cadherin, 5′-ATGCTGAGGATGATTGAGGTGGGT-3′ (forward) and 5′-CAAATGTGTTCAGCTCAGCCAGCA-3′ (reverse); β-catenin, 5′-TGCAGTTCGCCTTCACTATGGACT-3′ (forward) and 5′-GATTTGCGGGACAAAGGGCAAGAT-3′ (reverse); fibronectin, 5′-AAACTTGCATCTGGAGGCAAACCC-3′ (forward) and 5′-AGCTCTGATCAGCATGGACCACTT-3′ (reverse); vimentin, 5′-AGAACCTGCAGGAGGCAGAAGAAT-3′ (forward) and 5′-TTCCATTTCACGCATCTGGCGTTC-3′ (reverse); and β-actin, 5′-TGACGTGGACATCCGCAAAG-3′ (forward) and 5′-CTGGAAGGTGGACAGCGAGG-3′ (reverse).
7
Quantifying Antibiotic Resistance Genes
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Residual genomic DNA was completely removed by wipe Mix, and cDNA was synthesized via reverse transcription (Vazyme Biotech Co., Ltd, Nanjing, China). qPCR was performed on the CFX96 real-time PCR detection system (Bio-Rad Laboratories Co., Ltd, Hercules, CA, USA) using HiPer SYBR Premix EsTaq (Mei5 Biotechnology, Co., Ltd, Beijing, China). The mecA primer set was designed using primer Basic Local Alignment Search Tool (BLAST) for the region spanning 36–335 bp, and the housekeeping gene gryB was used as an internal standard for normalization (Table S1 in the supplemental material). The thermal cycling conditions were initial denaturation at 95°C for 2 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The experiment was repeated three times. Fold changes in mecA expression were illustrated using GraphPad Prism v8.0 (GraphPad Software Inc., San Diego, CA, USA). The mecA gene was amplified using the synthesized cDNA as template via conventional PCR. Products were sequenced using Sanger sequencing and the sequences were comparatively analyzed.
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