The single clone of MG1363, pGS/MG1363 and pGSMT/MG1363 was inoculated respectively in tube containing 5 mL of GM17 broth medium at 30 °C for 1 d. Cells of 1 mL were collected by centrifugation at 12,000 × g for 1 min at 4 °C. The pellets were lysed and the total RNA was extracted using HiPure Bacterial RNA Kit (Magen, Guangzhou, P.R. China). Total RNA of 1 μg was used as the template for cDNA synthesis primed with random hexamers (Bio-Rad, Hercules, CA, USA). The reaction mixture was incubated at 42 °C for 30 min followed by 5 min at 85 °C. The PCR conditions were as follows: one cycle at 94 °C for 3 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with an additional extension at 72 °C for 7 min. Three DNA fragments (mt, gst-sumo and 16s rRNA) were performed for each sample using different primers (Supplementary Table S1 ) and the PCR products were checked on 2% (w/t) agarose gel electrophoresis.
Hipure bacterial rna kit
Manufactured by Magen Biotechnology Co
Sourced in China
The HiPure Bacterial RNA Kit is a laboratory tool designed to efficiently isolate and purify high-quality bacterial RNA from various sample types. The kit utilizes a silica-based membrane technology to capture and elute the RNA, ensuring a reliable and consistent extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Random hexamers, by Bio-Rad (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Hiscript 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Modified bca protein assay kit, by Sangon (1 mentions)
Bacterial protein extraction kit, by Sangon (1 mentions)
Ds 11, by DeNovix (1 mentions)
Transstart tip green qpcr supermix, by Transgene (1 mentions)
Transscript all in one first strand cdna synthesis supermix, by Transgene (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Terminal transferase, by New England Biolabs (1 mentions)
Dnase on column kit, by Magen Biotechnology Co (1 mentions)
Goldenstar rt6 cdna synthesis mix, by Tsingke (1 mentions)
Quantstudio 7 flex, by Thermo Fisher Scientific (1 mentions)
Hipure total rna micro kit, by Magen Biotechnology Co (1 mentions)
Hiscript 2 one step qrt pcr sybr green kit, by Vazyme (1 mentions)
Master qpcr mix, by Tsingke (1 mentions)
7 protocols using hipure bacterial rna kit
1
RNA Extraction and cDNA Synthesis
2
Bacterial RNA Extraction and qRT-PCR Analysis
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Analysis of the potential predation DEGs was conducted by extracting total RNA from bacteria with HiPure Bacterial RNA Kit (Magen, Guangzhou, China) following the protocol as described by manufacturer. cDNA was synthesis with HiScript 1st Strand cDNA Synthesis Kit (Vazyme Biotech, Co., Ltd.#R111-01, China) after DNaseI treatment to remove genomic DNA contamination. It was then used as a template for qRT-PCR, which was performed with a PrimeScriptTM RT reagent Kit (Perfect Real Time) (Takara, Code No.: RR037A, Japan) on the Applied Bio-systems (QuantStudio 5, United States). The qRT-PCR response procedures adopt a two-step cycling protocol with a procedure of 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 60°C for 35 s. The data were analyzed by using software QuantStudioTM Design & Analysis Software v1.5.1. The 16S rRNA gene was selected as the internal control. Results obtained from three biological repeats with standard deviation (SD).
3
Bacterial Protein and RNA Extraction
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The culture was sampled every 6 h, and then concentrated or diluted to 1 (OD600). Protein was extracted using Bacterial Protein Extraction Kit (BS596, Sangon Biotech Co, Ltd, Shanghai, China) and quantified by a Modified BCA Protein Assay Kit (SK3051; Sangon Biotech Co, Ltd, Shanghai, China). Total RNA was extracted by HiPure Bacterial RNA Kit (R4181-01; Magen, China) and quantified by a spectrophotometer (DS-11; DeNovix, Wilmington, DE, USA).
4
Comparative RT-qPCR analysis of L. acidophilus
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Total RNA from L. acidophilus-0 and L. acidophilus-epsF was separately extracted using HiPure Bacterial RNA Kit (Magen, Guangzhou, China). cDNA was reverse-transcribed from equal quality (≤1μg) of the total RNA for RT-qPCR using TransScript® All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) following the manufacturer’s instructions.
RT-qPCR using TransStart® Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) and LightCycler® 96 (Roche, Basel, Switzerland) involved initial denaturation at 94 °C for 30 s and 45 cycles of denaturation at 94 °C for 5 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. 16S rRNA was the internal reference (Kim et al., 2015 (link)) for epsF. Primers No. 5–8 (Table 1 ) were designed for this step. The gene expression levels were estimated using the method (Livak & Schmittgen, 2001 (link)). Each experimental group was repeated three times.
RT-qPCR using TransStart® Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) and LightCycler® 96 (Roche, Basel, Switzerland) involved initial denaturation at 94 °C for 30 s and 45 cycles of denaturation at 94 °C for 5 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. 16S rRNA was the internal reference (Kim et al., 2015 (link)) for epsF. Primers No. 5–8 (
5
Transcriptome starts sites identification
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5′RACE was used to identify the transcription starts sites of repA and antisense RNA. The total RNA of strain BW25113/pHNSHP45 was extracted using HiPure Bacterial RNA Kit (Magen, China), and purified RNA samples were subjected to gDNA digestion using DNase On Column Kit (Magen, China) following the manufacturer's instructions. RNA integrity was checked on 1% agarose gels and quantified using NanoDrop (Thermo Scientific, USA). Reverse transcription was performed on 1 μg total RNA with special primers RepA-TSS or AS RNA-TSS using Goldenstar RT6 cDNA Synthesis Mix (TsingKe Biotech. China). The cDNA products were purified by MagPure cDNA Clean Up Kit (Magen, China). Purified cDNA was tailed by poly dC using terminal transferase (NEB, UK). Primer Race-TSS which contain poly dG and special primer RepA-TSS or AS RNA-TSS were used to perform PCR, and tailed cDNA as the template. Finally, the PCR products were cloned to pMD19T for Sanger sequencing.
6
Quantitative Analysis of Gene Expression in A. baumannii and Host Cells
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For each individual experiment, PBS or oligomer 3 (0 to 10 μg/ml) was added to different groups of samples (N = 3), and the samples were incubated as indicated vide supra. A. baumannii RNA and RNA of the NIH/3T3 cells or RAW 264.7 cells were extracted with the HiPure Bacterial RNA Kit (Magen, Shanghai) and the HiPure Total RNA Micro Kit (Magen, Shanghai), respectively, following the manufacturer’s instructions. The extracted RNA was converted to cDNA, and the qPCR was conducted in a real-time PCR system (QuantStudio 7 Flex, Thermo Fisher Scientific, USA) with the HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme, Nanjing). Gene expression was normalized to the expression of the housekeeping gene (16S rRNA for A. baumannii and 18S rRNA for NIH/3T3 or 18S rRNA for RAW 264.7). The primer sequences for each gene are listed in data file S1.
7
Quantifying Efflux Pump Gene Expression
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The total RNA of the recombinant strains was extracted using the TRIzol method with a HiPure Bacterial RNA Kit (Magen, China) according to the manufacturer’s instructions. A NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis were used to assess the concentration and quality of RNA, and cDNA was synthesized using an RNA Reverse Transcription Kit (TsingKe Biotech, China). qPCR was performed to measure the expression levels of efflux pump genes using the Tsingke Master qPCR Mix (TsingKe Biotech, China) with primers (Table S4) and the 16S rRNA gene as an internal reference. Based on a previous report, the 2△△–Ct method was used to analyze the relative expression values (49 (link)).
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