Genomic DNA was extracted for methylation analysis from cells after exposure to PS or OS for 6 hours. One gram of genomic DNA was modified with sodium bisulfite using the EpiMark Bisulfite kit (BioLabs) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) amplifications were carried out in a total volume of 25 μL by using 2× EasyTaq PCR SuperMix (TRANSGEN BIOTECH). The primer sets are listed in Table S1 . MSP reactions were subjected to initial incubation at 95 °C for 5 minutes, followed by 40 cycles of 95 °C for 30 seconds, and annealing at the appropriate temperature for 30 seconds and 72 °C for 30 seconds. Final extension was done by incubation at 72 °C for 5 minutes. MSP products were separated on 2% agarose gels and visualized after Gel-Red (Beyotime) staining.
Gel red
Manufactured by Beyotime
Sourced in China
Gel-Red is a nucleic acid gel stain used for visualization of DNA and RNA in agarose gels. It is a sensitive and stable fluorescent dye that emits bright red fluorescence upon binding to nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Primestar master mix, by Takara Bio (2 mentions)
Easytaq pcr supermix, by Transgene (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
Rhodamine b isothiocyanate, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Mncl2, by Maclin Biochemical Technology (1 mentions)
Pdms prepolymer sylgard 184, by Dow (1 mentions)
Mgso4, by New England Biolabs (1 mentions)
Dntps, by New England Biolabs (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Loading buffer, by Dingguo (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Dnase free proteinase k, by Beyotime (1 mentions)
Nanodrop nd 1000 uv visible spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Epimark bisulfite conversion kit, by New England Biolabs (1 mentions)
Taq dna polymerase, by Tiangen Biotech (1 mentions)
Taq dna polymerase, by GenStar (1 mentions)
Epijet bisulfite conversion kit, by Thermo Fisher Scientific (1 mentions)
Goscript system, by Promega (1 mentions)
17 protocols using gel red
1
DNA Methylation Analysis by Bisulfite PCR
2
Gel Retardation Assay for dsRNA Protection
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Gel retardation assay was used to investigate the protective effect of the nanoparticles on dsRNA. Briefly, dsRNA mPEI nanoparticles at a concentration of 1 μM dsRNA concentration were mixed with FBS (1 : 1, v/v) and then incubated at 37 °C for different time-points (0, 1, 2, 3, 6, 9, 12, 24, 36, and 48 h). After sampling, the samples were stored at −20 °C before the assay. The loading buffer of RNA (6×) was added to each sample, and then the sample was assayed by electrophoresis on 1% agarose gel containing 0.5 mg mL−1 Gelred (a special luminescent dye for staining RNA; Beyotime, Beijing, China). Electrophoresis was performed at 80 mV for 3 min, and subsequently 100 mV for 15 min. These resulting gels were photographed under the Imager Quant RTECL gel imaging system (Tanon, Shanghai, China). Free dsRNA was used as the control (dsRNA = 1 μM).
3
Oligonucleotide Synthesis and Characterization
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EGCG, rhodamine B isothiocyanate (Rho), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO). PLL (4224 Da) was purchased from Macklin (Shanghai, China). Rabbit monoclonal antibodies against Bcl-2 (catalog number: ab182858) and β-actin (catalog number: ab16039) were purchased from Abcam, Inc. (Cambridge, UK). Trizol was obtained from NCM Biotech. (Suzhou, China) and Master-mix with SYBR-green kit was obtained from Takara, Inc. (Japan). Commercial transfection reagent Lipofectamine 2000 (LPF) was purchased from Thermo Fisher Scientific, Inc (Shanghai, China) and TransExcellent-siRNA (TE) was purchased from Cenji Biotech. (Shanghai, China). RNase and RNase Inhibitor were obtained from Yeasen Biotech. (Shanghai, China). GelRed was obtained from Beyotime Biotech. (Shanghai, China).
ASO, Ps-ASO, and siRNA specifically targeting firefly luciferase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and prolyl hydroxylase-2 (PHD2), Bcl-2 DNAzyme, anti-microRNA-155 (anti-miR-155), scrambled ASO nonspecific to any gene (Sc-ASO), and ASO labeled with carboxyfluorescein at the 5′ end (ASO–FAM) were synthesized by Gene Pharma (Shanghai, China). The sequences of synthesized oligonucleotides were listed in Supplementary TableS1 . All the chemicals were used as received without further purification.
ASO, Ps-ASO, and siRNA specifically targeting firefly luciferase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and prolyl hydroxylase-2 (PHD2), Bcl-2 DNAzyme, anti-microRNA-155 (anti-miR-155), scrambled ASO nonspecific to any gene (Sc-ASO), and ASO labeled with carboxyfluorescein at the 5′ end (ASO–FAM) were synthesized by Gene Pharma (Shanghai, China). The sequences of synthesized oligonucleotides were listed in Supplementary Table
4
Comet Assay for DNA Damage
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Cells were seeded in 6-well plates. After treatment with NL-101 for 24 h, cells were harvested and resuspended at 1 × 105 cells/ml density in ice-cold PBS. And then they were combined with low-melting agarose (1 g/100 ml in 1 × Tris Base, Boric, acid, EDTA) at a ratio of 1 : 10 (v/v) and spread on the comet slide. The slides solidified for 10 min at 4°C and then submerged in alkaline lysis buffer at 4°C overnight. Then slides were subjected to electrophoresis at 1.0 V/cm for 30 min. The slides were placed in 50%, 70%, and 100% ethanol and dried at 37°C for 15 min, respectively. The slides were then stained with Gel-Red (Beyotime, Shanghai, China) for 20 min in dark. Images were acquired using a fluorescent microscope.
5
Rapid Detection of Foodborne Pathogens
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The LAMP reagent containing Bst 2.0 WarmStart DNA polymerase, 10 × isothermal amplification buffer, 100 mM MgSO4 and dNTPs, was purchased from New England BioLabs (Ipswich, MA, USA). Primers, plasmid DNA (pDNA) of E. coli O157:H7, Salmonella spp. and S. aureus, and agarose powder were obtained from Sangon (Shanghai, China). MnCl2 and Calcein (CAS No.: 154071–48-4, C30H26N2O13, 2,2′,2″,2″′-[(3′,6′-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]-2′,7′-diyl)bis (methanediylnitrilo)] tetraacetic acid) were purchased from Macklin (Shanghai, China) and Sigma-Aldrich (St. Louis, MO, USA), respectively. PDMS prepolymer (Sylgard 184) and curing agent were obtained from Dow Corning. Whatman filter paper grade 1 and Gel-Red was purchased from Beyotime Biotechnology (Shanghai, China). The Wizard genomic DNA purification kit was obtained from Promega (Madison, WI, USA). S. aureus real-time PCR kit was purchased from Zeye Biotechnology (Shanghai, China). A 100 bp DNA size marker was obtained from Sparkjade Biotechnology (Shandong, China). Loading buffer was purchased from Dingguo Biotechnology (Beijing, China). 50 × TAE buffer was purchased from YuanYe Biotechnology (Shanghai, China). Clinx Gel imaging system (Shanghai, China) was used to detect the target bands.
6
Comet Assay for DNA Damage
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After exposure, the cells were digested, resuspended in a culture medium, and then placed on ice. The sandwich agarose gels were made up of 0.65% normal-melting agarose that had been pre-coated on a slice and 0.65% low-melting agarose mixed with cells on normal-melting agarose. The cells were lysed in lysis buffer containing 1% Triton X-100 at 4°C for 1 h and then enzymatically hydrolyzed in lysis buffer containing 0.5 mg/ml DNase-free proteinase K (Beyotime, Shanghai, China) at 37°C for 2 h. Before electrophoresis, DNA in cells was unwind in ice-cold alkaline electrophoresis solution for 20 min and then electrophoresis at 20 V for 20 min. After electrophoresis, cells were neutralized in Tris buffer (0.4 M, pH 7.5) for 2 × 5 min. DNA “comets” were stained by Gel-red (Beyotime) and photographed by a fluorescence microscope (Zeiss, Oberkochen, German). Approximately 30 comets for each sample were calculated using the CASP 1.2.2 software (Krzysztof Konca, Wroclaw, Poland). Additional details can be found in a previously described protocol (32 (link)).
7
Total RNA Extraction with TRIzol Reagent
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Total RNA of each tissue was extracted with TRIzol Reagent (Cat No. 15596-026, Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. The concentration of RNA was determined by measuring the OD at 260 nm with the NanoDrop ND-1000 UV-Visible Spectrophotometer (Thermo Scientific, Carlsbad, MA, United States). The integrity was checked by separating the RNA on a 1% agarose gel and staining with Gel-Red (Cat No. D0139, Beyotime, Shanghai, China).
8
Methylation Analysis of ABCA1 and SOAT1
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Genomic DNA of RAW 264.7 was extracted by DNA extraction buffer (50 μl TE buffer, 450 μl STE buffer, 10 μl 20% SDS,10 μl protein K (10 mg/ml)). Bisulfite modification of DNA (1 μg) was performed with the EpiMark Bisulfite Conversion Kit (NEB, USA) according to the manufacturer’s instructions. MethPrimer (http://www.urogene.org/methprimer ) was used for determining the methylation status and design the primers of methylated or unmethylated sequences of ABCA1 and SOAT1. Polymerase chain reaction (PCR) amplifications were carried out in a total volume of 25 μl by using Taq DNA Polymerase (TIANGEN). MSP reactions were subjected to initial incubation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, and annealing at the appropriate temperature for 30 s and 72 °C for 30 s. Final extension was done by incubation at 72 °C for 7 min. MSP products were separated on 2% agarose gels and visualized after Gel-Red (Beyotime Biotechnology, Shanghai) staining. Primers for MSP are listed in Supplementary Table 1 .
9
Bisulfite-based DNA Methylation Analysis
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Genomic DNA was extracted from cells with DNA extraction buffers (50 μl TE buffer, 450 μl STE buffer,10 μl 20% SDS,10 μl protein K (10 mg/ml)) 39 (link). Bisulfite modification of DNA (1 μg) was performed by using the EpiJET Bisulfite Conversion Kit (Thermo scientific, K1461) according to the manufacturer's instructions. DNA was purified with a DNA Pure-Spin Kit (Vigorous, N009). MethPrimer platform was used for designing bisulfite-conversion-based MSP primers for ALDH2, ALDH3A1 and ALDH6A1. Polymerase chain reaction (PCR) amplifications were carried out in a total volume of 25 μl by using Taq DNA Polymerase (GenStar, A012-B101). The initial denaturation step of PCR amplification was 95 ℃ for 5 minutes, followed by 43 cycles of 95 ℃ for 30 seconds and 52 ℃ for 30 seconds and 72 ℃ for 30 seconds, then maintaining at 72 ℃ for 5 minutes, and last at 12 ℃ for infinite. MSP products were separated on 2% agarose gels and visualized after Gel-Red (Beyotime Biotechnology, Shanghai) staining. Primers are presented in Table S1 .
10
RNA Extraction and qPCR Analysis
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RNA was extracted using TRIzol (T9424, Sigma-Aldrich) and reverse transcribed with the GoScript system (A5001, Promega). RT-PCR was performed using PrimeSTAR Master Mix (R045A, Takara) according to the manufacturer’s instructions, including PCR control. Products were separated on a 2% agarose gel and visualized with GelRed (D0140, Beyotime). qPCR was conducted on a LightCycler 480 (Roche) with Platinum SYBR Green mix (11,744,500, Invitrogen). CircRNA, mRNA, and miRNA expression levels were normalized to GAPDH and U6, respectively, using the 2−ΔΔCt method for determining gene expression level [20 (link)]. Primer sequences are listed in Supplementary Table 1 .
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