Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA concentration and purity were measured using OPTIZEN NanoQ (Mecasys, Daejeon, Korea), and 1 µg/µL of total RNA was used to synthesize cDNA using AccuPower® RT-PCR Premix (Bioneer Inc., Daejeon, Korea). Specific primers for the sequences of interest (Table 5 ) were designed using the National Center for Biotechnology Information (NCBI) database. PCR was performed as follows: 95 °C for 5 min, followed by 30–35 cycles at 95 °C for 30 s, 55 to 61 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The PCR products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide, and photographed under UV illumination. Band intensities were quantified using ImageJ (National Institutes of Health, MD, USA).
Optizen nanoq
Manufactured by Mecasys
The OPTIZEN NanoQ is a compact and high-precision laboratory spectrophotometer designed for accurate absorbance measurements. It features a wide wavelength range, high-resolution optics, and reliable performance for diverse applications in research and quality control settings.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Accupower rt pcr premix, by Bioneer (2 mentions)
Kinetex c18 100a column, by Phenomenex (2 mentions)
Rezex roa organic acid h column, by Phenomenex (2 mentions)
Alliance 2796 separations system, by Waters Corporation (2 mentions)
1260 series hplc, by Agilent Technologies (2 mentions)
2996 photodiode array detector, by Waters Corporation (2 mentions)
Snapshot multiplex kit, by Thermo Fisher Scientific (1 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Genomic dna extraction kit, by Qiagen (1 mentions)
Ripa lysis buffer system, by Santa Cruz Biotechnology (1 mentions)
Nb600 503, by Novus Biologicals (1 mentions)
8 protocols using optizen nanoq
1
Quantitative Gene Expression Analysis by RT-PCR
2
HPLC Analysis of Fermentation Metabolites
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The glucose, fructose, glycerol, acetate, and ethanol concentrations of the hydrolysis and fermentation samples were analyzed using HPLC (HPLC 1260 series, Agilent Technologies, USA) equipped with a Rezex-ROA Organic Acid H+ column (8%, 150 mm × 4.6 mm; Phenomenex Inc., USA). The analytes were eluted with 0.005 N H2SO4 at 0.6 ml/min and 50°C, as previously described [20 (link)].
Sulforaphane was analyzed using HPLC (Waters, Alliance 2796 Separations System), with a 2996 photodiode array detector (Waters) connected to a Kinetex C18 100A column (5 μm, 250 mm × 4.6 mm: Phenomenex Inc.). The filtrate was analyzed by acetonitrile/water (3:7, v/v) isocratic elution at a flow rate of 0.6 ml/min. A UV/Vis spectrophotometer (Optizen NanoQ, Mecasys Co., Korea) at a wavelength of 205 nm was used to detect sulforaphane.
Sulforaphane was analyzed using HPLC (Waters, Alliance 2796 Separations System), with a 2996 photodiode array detector (Waters) connected to a Kinetex C18 100A column (5 μm, 250 mm × 4.6 mm: Phenomenex Inc.). The filtrate was analyzed by acetonitrile/water (3:7, v/v) isocratic elution at a flow rate of 0.6 ml/min. A UV/Vis spectrophotometer (Optizen NanoQ, Mecasys Co., Korea) at a wavelength of 205 nm was used to detect sulforaphane.
3
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA concentration and purity were measured with an OPTIZEN NanoQ (Mecasys, Daejeon, Korea). Subsequently, 1 µg/µL of total RNA and AccuPower® RT-PCR Premix (Bioneer Inc., Daejeon, Korea) was used to synthesize cDNA. Specific primers for the sequences of interest (Table 1 , Table 2 and Table 3 ) were designed using the NCBI database. PCR was carried out at 95 °C for 5 min, followed by 30–35 cycles comprising the following steps: 95 °C for 30 s, 55–61 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The PCR products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide (EtBr), and photographed under UV illumination. Band intensities were quantified while using the ImageJ software (version 1.47, National Institutes of Health, Bethesda, MD, USA).
4
Sheep SNP Genotyping Assay
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One-hundred eleven matched pairs (case-control) were identified among the retrospective cohorts and genetic analyses were performed on these sheep. Genomic DNA was extracted from whole blood with EDTA using commercial kits (General Biotechnology Co., Ltd., Seoul, Korea). DNA quality was confirmed via spectrophotometer (Optizen-NanoQ, Mecasys Co., Ltd, Daejeon, Korea). A multiplex Single Nucleotide Primer Extension (SNuPE) assay was designed for genotyping of the samples. Briefly, a multiplex Polymerase Chain Reaction (PCR) was used to amplify the targeted regions of DNA. The extension primers without fluorescent dye were designed in a specific length for each SNP and was expected to bind to preceding nucleotide of the targeted SNP. The 'T' tails were added to ensure that the extension primers were of different lengths for each SNP. Amplification and extension primers and rs numbers of the analyzed SNPs are provided in Supporting Information Table S1. The SNuPE reaction was performed using SNaPshot TM Multiplex Kit (Thermo Fisher Scientific Inc., USA) and capillary electrophoresis was used for fragment analysis protocol on Applied Biosystems 3500 genetic analyzer. To validate SNuPE genotyping assay, approximately 5-10% of the samples for each SNP were directly sequenced and the results were in 100% concordance.
5
Genetic Profiling of Ovis aries PAPPA2
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Genomic DNA was extracted from whole blood using a commercial spin column kit (General Biotechnology Co., Ltd., Seoul, Korea). DNA quality and quantity were determined by a spectrophotometer (Optizen-NanoQ, Mecasys Co., Ltd, Daejeon, Korea). The polymorphic regions of the Ovis aries PAPPA2 gene (OAR 3.1) were selected as target regions. Oligonucleotide primers were designed to amplify parts of PAPPA2 5'UTR, exon 1, exon 2, exon 5, and exon 7 (Supplementary Table S1). A Polymerase Chain Reaction (PCR) was employed to amplify the target regions. PCR products were eluted in 2% agarose gel under ultraviolet (UV). The chain termination reaction was performed using BigDye™ Terminator v3. 1 Cycle Sequencing Kit (Thermo Fisher Scienti c Inc., USA) and capillary electrophoresis was conducted using Applied Biosystems 3500 genetic analyzer platform.
6
Extraction of Lactobacillus mucosae LM1 Genomic DNA
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L. mucosae LM1 was grown in 5 mL de Man-Rogosa-Sharpe (MRS) broth (Difco, Pontde-Claix, France) at 37°C under static conditions for 24 h. Genomic DNA was isolated using the Qiagen Genomic DNA Extraction Kit (Hilden, Germany) according to the protocol provided by the manufacturer. DNA quality was verified by agarose gel electrophoresis, and DNA concentration and purity (A260/A280) were measured using an Optizen Nano Q (Mecasys, Daejeon, Korea).
7
Protein Isolation from Tissue Samples
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For protein isolation, tissue powders in each replicate were homogenized by a Daihan 15D tissue homogenizer (27,000 rpm) in 400 μL of RIPA Lysis Buffer (including 2 μL of PMSF, 2 μL of a sodium orthovanadate, and 2 μL of a protease inhibitor cocktail) (RIPA Lysis Buffer System, sc-24948; Santa Cruz, CA, USA) under ice-cold conditions. The lysate was centrifuged at 14,000 g for 20 minutes and in the range of 2.13–3.17 mg/mL of total protein was detected by the protein A280 method with a micro-volume spectrophotometer (Optizen Nano Q; Mecasys). The protein level was measured using a Western blot assay and SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) electrophoresis, as described by Delen et al. [15 (link)] with two primary antibodies (DDR1 [NBP1-33134; Novus Biologicals, Littleton, CO, USA] and DDR2 [NBP2-14927; Novus Biologicals]) and normalized with a beta-actin antibody (NB600-503; Novus Biologicals). Protein quantities were determined with the ProteinQuant software and given as relative density.
8
HPLC Analysis of Fermentation Metabolites
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The glucose, fructose, glycerol, acetate, and ethanol concentrations of the hydrolysis and fermentation samples were analyzed using HPLC (HPLC 1260 series, Agilent Technologies, USA) equipped with a Rezex-ROA Organic Acid H+ column (8%, 150 mm × 4.6 mm; Phenomenex Inc., USA). The analytes were eluted with 0.005 N H2SO4 at 0.6 ml/min and 50°C, as previously described [20 (link)].
Sulforaphane was analyzed using HPLC (Waters, Alliance 2796 Separations System), with a 2996 photodiode array detector (Waters) connected to a Kinetex C18 100A column (5 μm, 250 mm × 4.6 mm: Phenomenex Inc.). The filtrate was analyzed by acetonitrile/water (3:7, v/v) isocratic elution at a flow rate of 0.6 ml/min. A UV/Vis spectrophotometer (Optizen NanoQ, Mecasys Co., Korea) at a wavelength of 205 nm was used to detect sulforaphane.
Sulforaphane was analyzed using HPLC (Waters, Alliance 2796 Separations System), with a 2996 photodiode array detector (Waters) connected to a Kinetex C18 100A column (5 μm, 250 mm × 4.6 mm: Phenomenex Inc.). The filtrate was analyzed by acetonitrile/water (3:7, v/v) isocratic elution at a flow rate of 0.6 ml/min. A UV/Vis spectrophotometer (Optizen NanoQ, Mecasys Co., Korea) at a wavelength of 205 nm was used to detect sulforaphane.
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