Gradient ultracentrifugation was used to further purify selected samples enriched in sEVs either by TFF or dUC. Samples containing about 200 μg of sEVs (expressed in protein content and measured by BCA) were concentrated by ultracentrifugation at 110,000 × g for 2 h at 4°C using a Ti70 rotor. The resulting pellets were homogenized in 10 mM Tris‐HCL pH 8.6 buffer in a final volume of 50 μl. Samples were vigorously mixed for 20 min to disaggregate vesicles. A 50% (w/v) iodixanol working solution was prepared by diluting OptiPrep (Merck) according to the manufacturer's instruction. A discontinuous gradient containing 1.5 ml of 50%, 30% and 10% gradient cushions was prepared and the samples containing the EVs of interest were layered on top of the gradient. Ultracentrifugation was carried out at 110,000 x g for 24 h at 4°C using an SW55Ti rotor. Ten fractions of 500 μl each were collected from the top of the tubes. The percentage of iodixanol in each fraction was measured using a UV spectrophotometer (Nanodrop 200, Thermo Scientific) at 340 nm, from which the density was calculated according to the manufacturer's method. Protein concentration in each fraction was measured by micro‐BCA (Thermo Scientific) using a UV spectrophotometer (Nanodrop 200, Thermo Scientific) at 562 nm.
Nanodrop 200
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop 200 is a spectrophotometer designed for the quantification and qualification of nucleic acids and proteins. It utilizes a small sample volume of 1-2 microliters to provide accurate and reproducible measurements.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (6 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (5 mentions)
Superscript 2, by Thermo Fisher Scientific (5 mentions)
Lightcycler 480 instrument 2, by Roche (5 mentions)
Random hexamer, by Thermo Fisher Scientific (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Perfecta sybr green fastmix, by Quanta Biosciences (3 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Optiprep, by Merck Group (1 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
Kasp genotyping platform, by LGC (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Taqman primer, by Thermo Fisher Scientific (1 mentions)
Quantstudio 7 system, by Thermo Fisher Scientific (1 mentions)
Genomic mini kit, by A&A Biotechnology (1 mentions)
Rnalater, by Merck Group (1 mentions)
Trizol method, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Sensimix sybr fluorescein kit, by Meridian Bioscience (1 mentions)
28 protocols using nanodrop 200
1
Gradient Ultracentrifugation for sEV Purification
2
RNA Extraction and Quantitative PCR
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The total RNA from different cell lines were extracted using RNeasy Mini Kit (Qiagen, Germany) according to standard protocol. The RNA concentration was measured by NanoDrop 200 (Thermo Scientific, USA). Following standard protocol, first-strand complementary DNA (cDNA) was synthesized using Superscript II and Random Hexamer primer (Invitrogen, USA). Master Mix LightCycler 480 SYBR Green I (Roche, Switzerland) was used to complete the quantitative reaction using LightCycler 480 Instrument II (Roche, Switzerland). A constant melting temperature and cycle number for amplification were maintained for each reaction. The specific target detection was evaluated by melting curve analysis. Here, GAPDH was used as the housekeeping gene, and relative expression was calculated by 2-ΔΔCt method. The following primer sequences were used in this study: GAPDH, forward: 5′-TGCCATCAATGACCCCTTC-3′ and reverse, 5′-CATCGCCCCACTTGATTTTG-3′; SNHG8, forward: 5′-CCCGAGAACCGTCAGTTTGA-3′ and reverse, 5′-ACACCCGTTTCCCCAACTAC-3′; HPRT1, forward: 5′-TGC TCGAGATGTGATGAAGG-3′, and reverse, 5′-TCCCCTGTT GACTGGTCATT-3′. All primers were purchased from IDT (United States).
3
Validating SNP Conversion from Array to KASP Genotyping
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To validate the conversion of selected SNPs from the Illumina 90 k array to the Kompetitive Allele-Specific PCR (KASP) genotyping platform (LGC Genomics, UK), DNAs were extracted from the eight MAGIC founder varieties. Marker co-dominance was investigated using a 50:50 mix of DNAs from two varieties known from the 90 k SNP dataset to contrast for allele call. Additionally, DNA was extracted from a panel of 48 UK varieties that post-date the AM panel, released to the AHDB Recommended List between 2009 and 2017 (Supplementary Table 2). All DNAs were extracted from two week old leaves using a modified Tanksley protocol (Fulton et al. 1995 ), and concentration was determined using a Nanodrop 200 spectrophotometer (Thermo Scientific). DNA sequences flanking each targeted SNP were used to design KASP primers using the software PolyMarker (Ramirez-Gonzalez et al. 2015 (link)). Primers were synthesised by Sigma-Aldrich (Cambridge, UK) and KASP genotyping undertaken as described by Cockram et al. (2015 ). The results were visualised using SNP Viewer v.1.99 (https://lgcgenomics.com/ ). The subset of plants that post-dated the AM panel were grown and phenotyped for ToxB sensitivity and genotyped with the KASP marker, following the methods described above.
4
Isolation of cffDNA and Maternal DNA
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Venous blood samples were taken from pregnant women and their husbands, and collected in a 3 mL EDTA tube. The tube was centrifuged at 1,200 × g for 10 min at 4°C to separate the plasma from the red blood cells. The upper layer, which was the plasma, was transferred to a 1.5 mL tube and centrifuged further using a Norgren Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit (Cat. No. 55100; Norgen Biotek, Canada) according to the manufacturer’s protocol. The plasma was centrifuged at 2,400 × g for 20 min at 4°C, as described previously (12 (link)), to isolate the cffDNA. Furthermore, the buffy coat layer was taken for isolation of maternal DNA using the homebrew method. The concentration of DNA was measured by spectrophotometer (Nanodrop 200; Thermo Scientific). As a positive control, we used archived DNA samples from patients with β-thalassemia major with known mutations (Sanger Sequencing), and this positive control was rerun using pyrosequencing to confirm the mutation.
5
Quantitative RT-PCR Analysis of Gene Expression
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Frozen lung tissue was ground to a fine powder using a mortar and pestle. RNA from isolated alveolar macrophages or lung tissue were purified using the RNeasy mini kit as per manufacturers instruction (Qiagen, GMH Germany). The NanoDrop200 (Thermo Fischer Scientific, United States) was used to determine the concentration and purity of extracted RNA and the High-Capacity RNA to cDNA kit (Thermo Fischer Scientific, United States) was used to generate complementary DNA (cDNA). RT-qPCR was performed using the QuantStudio 7 system (Thermo Fischer Scientific, United States), TaqMan primers and the Taqman Fast Advanced Master Mix, as per manufacturer’s instructions. The relative expression of each gene was normalised against the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) and determined using the ΔΔCt value method, as previously described (Wang et al., 2018 (link); Wang et al., 2019 (link)).
6
Quantification of Colon Cell RNA Levels
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Total RNA from the colon cells was extracted using RNeasy mini kit (Qiagen, Germany) according to manufacturer guidelines. The RNA concentration was measured by NanoDrop 200 (Thermo Fisher Scientific, United States). Following standard protocol, first-strand cDNA was synthesized using Superscript II and Random Hexamer (Invitrogen, United States). Master Mix LightCycler 480 SYBR Green I (Roche, Switzerland) was used to complete the quantitative reaction using LightCycler 480 Instrument II (Roche, Switzerland). Here, GAPDH was considered as the housekeeping gene, and relative expression was calculated by 2–ΔΔCt method. The following self-designed primer sequences were used in this study: GAPDH, forward: 5′-TGCCATCAATGACCCCTTC-3′ and reverse, 5′-CATCGCCCCACTTGATTTTG-3′; CASC9, forward: 5′-TTGGTCAGCCACATTCATGGT-3′ and reverse, 5′-AGTGCCAATGACTCTCCAGC-3′; HPRT1, forward: 5′-TGC TCGAGATGTGATGAAGG-3′, and reverse, 5′-TCCCCTGTT GACTGGTCATT-3′. All primers were purchased from IDT (United States).
7
Gut DNA methylation, oxidation, and damage
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DNA was used for measuring the following: DNA global methylation, AP sites and oxidative stress biomarker 8-hydroxy-2′-deoxyguanosine (8-OHdG). DNA was isolated using Genomic Mini Kit (A&A Biotechnology, Gdansk, Poland). Briefly, RNA Later (Merck) was removed with a tube from the gut. Then tissue was homogenized, twice washed with Tris buffer (Genomic Mini Kit, A&A Biotechnology) and then all the steps in the protocol were followed. DNA was suspended in Tris buffer (100 µL), and DNA quality and concentration were measured using Nano Drop200 (Thermo Fisher Scientific, Waltham, MA USA). DNA was stored at −80 °C for future analysis.
8
Real-Time qRT-PCR Gene Expression Analysis
9
Efficient Transformation of E. coli with Plasmid
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One Shot MAX Efficiency DH5α-T1R competent cells (Invitrogen, Massachusetts, USA) were incubated with 1 µl of plasmid on ice for 30 min. These cells were then heat shocked at 42 °C for 30 s, returned to ice for 2 min, and grown in 950 µl SOC media (Invitrogen, Massachusetts, USA) at 37 °C with shaking for 1 h. Resulting transformed Escherichia coli cells were plated on LB agar with ampicillin (100 µg/ml; Sigma Aldrich, Missouri, USA) and grown over night at 37 °C. Colonies were then grown 8 h in 10 ml LB medium with ampicillin (100 µg/ml) followed by growing 400 ml to extract plasmids. Transfection-grade plasmid DNA was obtained using the NucleoBond Xtra Midi or Maxi kits (Macherey–Nagel, Düren, Germany). Final DNA product was assessed for quantity and quality using NanoDrop 200- (Thermo Fisher Scientific, Massachusetts, USA).
10
DNA Concentration by UV-Vis Spectroscopy
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The DNA concentration was determined by a UV–vis Spectrometer based on the extinction at 260 nm (NanoDrop 200, Thermo Scientific), whereby deionized water was used as blank.
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