Fecal samples were collected from standard cages where animals were housed during the experiments on days 1, 14, 28, 42, 56, 70 and 84 and stored at −80 °C until DNA extraction. Fecal samples from single experimental group were pooled, and Nucleic acids were extracted from 250 mg of sample using PowerSoil® DNA Isolation Kit (MoBio Laboratories, Inc., Carisbad, CA, USA) according to the manufacturer’s recommendations. DNA quality was checked using a Nanodrop 100™ (NanoDrop Technologies, Wilmington, DE, USA). The V3-V4 hypervariable regions of the 16S rRNA gene were amplified using the universal primers 341F and 785R, and then sequenced on an Illumina MiSeq (Illumina, San Diego, CA, USA) platform as per the manufacturer’s instructions, at Wellmicro Srl (Bologna, Italy). All sequence data were processed using a pipeline combining PANDASeq [22 (link)] and QIIME 2 [23 (link)]. Briefly, quality-filtered reads were binned into Amplicon Sequence Variants (ASVs) using DADA2 [24 (link)]. Singletons and chimeras were removed. Taxonomy assignment was performed using the VSEARCH algorithm [25 (link)] and the Greengenes database (May 2013 release). Alpha diversity was computed using the inverse Simpson index. Beta diversity was estimated by calculating Bray–Curtis distances between genus-level microbial profiles, which were then used as input for Principal Coordinates Analysis (PCoA).
Nanodrop 100
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nanodrop 100 is a spectrophotometer designed to measure the concentration and purity of nucleic acid and protein samples. It uses a minimal sample size of 1-2 microliters to determine the absorbance or optical density of a sample across a wide range of wavelengths.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Trizol reagent, by Merck Group (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy plus micro kit, by Qiagen (2 mentions)
Hiscript q rt supermix, by Vazyme (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Powersoil dna isolation kit, by Qiagen (1 mentions)
Ribopure tm of high quality total rna kit, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Genomestudio genotyping module, by Illumina (1 mentions)
Lightcycler apparatus, by Roche (1 mentions)
Sensifast sybr no rox kit, by Meridian Bioscience (1 mentions)
Tissuelyzer, by Qiagen (1 mentions)
Intron spanning primers, by Merck Group (1 mentions)
Lna hydrolysis probes, by Roche (1 mentions)
Lightcycler taqman master, by Roche (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
27 protocols using nanodrop 100
1
Fecal Microbiome Analysis Pipeline
2
Transcriptomic Analysis of Skeletal Muscle
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A total of 24 animals were randomly chosen among the available ones, assuring the representation of all available litters, and used to perform transcriptomic analysis (6 animals of each genetic type at each studied age). Total RNA was extracted from 50-100mg samples of LD muscle using the RiboPure TM of High Quality total RNA kit (Ambion, Austin, TX, USA) following the manufacturer’s recommendations. RNA was quantified using a NanoDrop-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The quality of the RNA was evaluated using the RNA Integrity Number (RIN) value from the Agilent 2100 Bioanalyzer device (Agilent technologies, Santa Clara, CA, USA). The RIN values ranged from 7.8 to 9.8.
3
Pig Genomic DNA Extraction and Genotyping
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Genomic DNA was isolated from the pieces of pig ear tissue using the DNA Extran-2 kit (Syntol, Russia) based on the Proteinase K lysis and isopropanol precipitation technique according to the manufacturer’s instructions. About 10 mg of tissue was used for DNA isolation for each sample.
The integrity of the obtained DNA samples was assessed by electrophoresis in 1% agarose gel; the quality of the samples was estimated based on the OD 260/280 and OD 260/230 ratios obtained using NanoDrop100 (Thermo Fisher Scientific, United States), and DNA quantity in the sample was estimated using the Qubit fluorometer (Invitrogen, United States).
Genotypes for all animals included in the analysis were obtained using the GeneSeek-Neogen GGP Porcine HD (INF Porcine 80K) BeadChip (Illumina, United States) consisting of approximately 70,000 SNPs evenly distributed throughout the pig genome at the average distance of 25 kbp. Raw fluorescence intensity data were processes with the aid of the GenomeStudio Genotyping module (Illumina, United States). The threshold genome calling significance level (GC score) was set at 20%, the other settings were as default. Genotypes quality control was performed using the PLINK 2.0 software (Purcell et al., 2007 (link)).
The integrity of the obtained DNA samples was assessed by electrophoresis in 1% agarose gel; the quality of the samples was estimated based on the OD 260/280 and OD 260/230 ratios obtained using NanoDrop100 (Thermo Fisher Scientific, United States), and DNA quantity in the sample was estimated using the Qubit fluorometer (Invitrogen, United States).
Genotypes for all animals included in the analysis were obtained using the GeneSeek-Neogen GGP Porcine HD (INF Porcine 80K) BeadChip (Illumina, United States) consisting of approximately 70,000 SNPs evenly distributed throughout the pig genome at the average distance of 25 kbp. Raw fluorescence intensity data were processes with the aid of the GenomeStudio Genotyping module (Illumina, United States). The threshold genome calling significance level (GC score) was set at 20%, the other settings were as default. Genotypes quality control was performed using the PLINK 2.0 software (Purcell et al., 2007 (link)).
4
Gene Expression Analysis of Cells Treated with Fruti
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Human MCF-7, HepG2 and Fibroblast cells were seeded in 12 wells plate. After reaching confluence, cells were incubated with vehicle (0) or fruti at 10, 25, 50, 75 or 100 µM for 24 h (concentrations previously chosen by WST1 assay screening). Total RNA was extracted from cultured cells using Trizol reagent (Sigma-Aldrich, St. Louis, Missouri, USA) and measured by Nanodrop 100 (Thermo Scientific, Waltham, Massachusetts, USA). Complementary DNA was synthesized from 2 µg total RNA with an oligo-dT, random hexamers primers and deoxythymidine oligomer, Ribolock RNAse inhibitor and RevertAid reverse transcriptase. Real-time polymerase chain reaction was performed in a Lightcycler apparatus (Roche, Mannheim, Germany) using the sensiFAST SYBR No-ROX kit (Bioline, London, UK). Initial fluorescent values were calculated by LinRegPCR3 (version 2013.0; Academic Medical Center, Amsterdam, The Netherlands). Primer sequences were described in Supplementary Table S2 . The most stable reference genes were calculated using geNorm40 (link). Transcript levels were normalized to housekeeping genes ratio GAPDH and Cyclophilin (CyP) for MCF-7 and Fibroblasts cells, and the genes ratio HPRT and Actin for HepG2 cells.
5
Genomic DNA Extraction from Porcine PBMCs
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Genomic DNA from 305 sows was extracted from peripheral blood mononuclear cells (PBMCs) by standard protocols as described in [28 (link)]. First, PBMCs were incubated with proteinase K and lysis buffer and then treated with RNaseI prior to be subjected to phenol/chloroform purification and isopropanol precipitation as in [28 (link)]. DNA concentration and purity were assessed by Nanodrop-100 (Thermo Fisher Scientific, Waltham, MA, USA).
6
Total RNA Isolation from Rat Liver
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Total RNA was purified from frozen rat liver using an RNeasy Mini Kit (Qiagen Ltd. Manchester, UK) according to manufacturer's specifications. Approximately 50 mg tissue, 1 mL QIAzol and a 5 mm metal bead were added to a 2 mL microcentrifuge tube. The samples were lysed and homogenised in a TissueLyzer (Qiagen Ltd. Manchester, UK), and subsequently incubated at room temperature for 5 min. Chloroform (200 µL) was added to each sample, vortexed thoroughly and incubated at room temperature for 3 min. The samples were centrifuged at 4 °C and 12,000×g for 15 min. The aqueous (upper) fraction containing RNA was transferred to a new tube and 1 volume of 70% ethanol was added. The samples were vortexed, added to spin columns and centrifuged at 8000×g for 15 s to bind RNA to the membrane, with the remainder of the procedure continuing as described in the RNeasy RNA purification kit. RNA concentration was quantified at 260 nm using a NanoDrop 100 (Thermo Fisher Scientific Inc., Paisley, UK).
7
Total RNA Isolation and Quantification
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Total RNA was isolated from the infected cells using RNeasy mini-kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with subsequent DNaseI treatment on the column. The amount of RNA concentration was determined by spectrometry using a Nanodrop 100 (Thermo Scientific, DE, USA).
8
Quantification of Activation Genes in U937 Cells
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To assess the expression of activation-related gene transcripts, RNA was extracted from U937 cells according to the manufacturer’s recommendations. Electrophoresis in 1% agarose gel was used to assess RNA integrity, and concentration and purity by absorbance 268/280 nm in a Nanodrop 100 (Thermo, USA). cDNA was generated using 250 ng of total RNA, random primers, and the Transcriptor first strand cDNA synthesis kit (Roche Cat. 04379012001, Mannheim Germany). Real time PCR was conducted with LNA hydrolysis probes from the Universal Probe Library Roche (UPL) (Roche, Cat 04683633001, Mannheim Germany), and intron spanning primers from Sigma. TLR2 (NM_003264.3) F 5′-CGTTCTCTCAGGTGACTGCTC-3′, R‘3-TCTCCTTTGGATCCTGCTTG-’5, UPL probe 14; CASP3 (NM_004346.3) F 5′-CTGGTTTTCGGTGGGTGT-3′, R 3′-CCACTGAGTTTTCAGTGTTCTCC-5′ UPL probe 34; CD11B (NM_000632.3) F 5′-GGCATCCGCAAAGTGGTA-3′, R 3′-GGATCTTAAAGGCATTCTTTCG-5′ UPL probe 9; and GADPH (NM_002046.3) F 5′-AGCCACATCGCTCAGACAC-3′, R 3′-GCCCAATACGACCAAATCC-5′ UPL probe 60.
One μL of each cDNA was amplified with 400 nM of primers, 100 nM of UPL probe, with the LightCycler TaqMan® Master (Roche, Cat. 04535286001) followed by 45 cycles of 95° 10 s, 60° 60 s, and 72° 1 s. Reference gene GAPDH was used for relative quantification, according to the 2−ΔΔCt method, using the non-stimulated cells as calibrator sample.
One μL of each cDNA was amplified with 400 nM of primers, 100 nM of UPL probe, with the LightCycler TaqMan® Master (Roche, Cat. 04535286001) followed by 45 cycles of 95° 10 s, 60° 60 s, and 72° 1 s. Reference gene GAPDH was used for relative quantification, according to the 2−ΔΔCt method, using the non-stimulated cells as calibrator sample.
9
Plasmid DNA Extraction from Sewage
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The plasmid DNA yields from the sewage samples were evaluated by using gel electrophoresis and a Qubit dsDNA BR assay kit on a Qubit 2.0 fluorometer (Invitrogen). Plasmid DNA purity was measured and validated at absorbance ratios of 260/280 and 260/230 using a NanoDrop 100 (Thermo Fisher). During pilot experiments aimed at protocol development and plasmid DNA enrichment, we also assessed the quality of our plasmid DNA preparations using a 2100 Bioanalyzer (Agilent).
10
Total RNA Isolation and Quality Assessment
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Total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to the manufacturer’s protocol, and then the genomic DNA was purified with RNase-free DNase I (Promega). The total RNA concentration was assessed using a NanoDrop-100 (Thermo Scientific, Wilmington, USA) and its quality was tested using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). All samples had A260/A280 and A260/A230 ratios of 2.13–2.23, RIN (RNA Integrity Number) value above 8.5, and 23 S/16 S values above 1.6 except for one.
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