For microarray hybridization, total RNA was isolated by homogenizing tissue samples of hypothalami using TissueLyser II (QIAGEN, Crawley, UK) and purified using a DNA-free RNA isolation kit (RNAqueous-4PCR kit; Ambion, Austin, TX, USA) according to the manufacturer’s instructions. The integrity and quantity of total RNA were assessed with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) before the microarray experiments. Its quality was assessed by agarose gel electrophoresis using 1% gels. RNA samples were first amplified for array analysis using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX, USA) in accordance with the manufacturer’s instructions. Briefly, 500 ng of total RNA was used to prepare labelled cRNA with overnight incubation according to the manufacturer’s protocol. The quality and quantity of the labelled cRNA were monitored using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). Amplified cRNA (1.5 ng) was hybridized on MouseWG-6 v2 Expression BeadChip arrays, containing more than 45,200 well-annotated Ref transcripts in mice, according to the manufacturer’s standard protocol. The array were then scanned on a BeadArray Reader (BeadStation 500 G Instrument, Illumina Inc.), and Spot images were identified and quantified by the Genome Studio software v1.0.2. (Illumina Inc.).
Nanodrop 2000 spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, China, United Kingdom, Japan, Canada, France, Italy, Australia, Spain, Denmark, Switzerland, Singapore, Poland, Ireland, Belgium, Netherlands, Lithuania, Austria, Brazil
The NanoDrop 2000 spectrophotometer is an instrument designed to measure the concentration and purity of a wide range of biomolecular samples, including nucleic acids and proteins. It utilizes a unique sample retention system that requires only 1-2 microliters of sample to perform the analysis.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2213 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (868 mentions)
Rneasy mini kit, by Qiagen (711 mentions)
Trizol, by Thermo Fisher Scientific (528 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (377 mentions)
Primescript rt reagent kit, by Takara Bio (330 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (224 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (217 mentions)
2100 bioanalyzer, by Agilent Technologies (212 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (206 mentions)
Iscript cdna synthesis kit, by Bio-Rad (184 mentions)
Mirneasy mini kit, by Qiagen (184 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (183 mentions)
Dnase 1, by Thermo Fisher Scientific (170 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (169 mentions)
Qiaamp dna mini kit, by Qiagen (137 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (131 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (125 mentions)
Qiaamp dna blood mini kit, by Qiagen (123 mentions)
Dneasy blood and tissue kit, by Qiagen (117 mentions)
6 577 protocols using nanodrop 2000 spectrophotometer
1
Microarray Analysis of Hypothalamic RNA
2
DNA and RNA Extraction for Taihang Chickens
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DNA extraction kits (Tiangen, Beijing, China) were used to extract DNA from the blood samples of 800 Taihang chickens. extracted DNA samples were tested for concentration and quality using a NanoDrop2000 spectrophotometer (Thermo, Shanghai, China) and 1.5% agarose gel electrophoresis imaging. After evaluation, the qualified DNA samples were sent to Novogene Technology Company for sequencing and genotyping.
Total RNA extraction was performed using the Trizol kit (Tiangen, Beijing, China). After testing the quality and concentration of RNA samples with a Nanodrop 2000 spectrophotometer (Thermo, Shanghai, China), the qualified samples were reverse transcribed into cDNA using a reverse transcription kit (Takara Bio, Japan) and stored at −20°C.
Total RNA extraction was performed using the Trizol kit (Tiangen, Beijing, China). After testing the quality and concentration of RNA samples with a Nanodrop 2000 spectrophotometer (Thermo, Shanghai, China), the qualified samples were reverse transcribed into cDNA using a reverse transcription kit (Takara Bio, Japan) and stored at −20°C.
3
RT-PCR Analysis of Ewing Sarcoma Tumors
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Archival frozen tumor specimens and RD-ES cell line served as positive controls for nested RT-PCR experiments. Frozen tumor samples from 5 untreated ES patients were obtained from KU Leuven and University Hospitals, Belgium (4 primary tumors and 1 metastatic tumor; 4 males and 1 female; median age 25 years, range 4–43 years). Total RNA from the frozen tumor specimens was extracted using miRNeasy kit (Qiagen) including DNase treatment using RNease-free DNase set (Qiagen) according to the manufacturer’s protocol. RNA quantity was determined using NanoDrop 2000 Spectrophotometer (Thermo Scientific), and the quality was evaluated using Bio-Rad Experion RNA StdSens Analysis system (Bio-Rad, Hercules, CA, USA). RD-ES cell line carrying EWS–FLI1 type 2 translocation was purchased from CLS Cell Lines Service GmbH. Total RNA was extracted from RD-ES cells using RNeasy kit (Qiagen) according to the manufacturer’s protocol. RNA quantity and quality were evaluated using NanoDrop 2000 Spectrophotometer (Thermo Scientific) and FlashGel system (Lonza), respectively.
4
DNA Quantification and Purity Assessment
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DNA concentration (ng/μL) was determined using a Qubit 2.0TM Fluorometer with dsDNA BR Assay KitTM for Qubit and a NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The DNA purity (260/280 and 260/230 ratios) was assessed by NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The two extraction protocols determined the concentration and purity from swabs and peripheral whole blood samples.
5
Wheat Cultivar Transcriptome Analysis
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Surface sterilized seeds of 12 wheat cultivars were grown in germination papers configured to roll-ups under well-watered and water-limited conditions. Two weeks after sowing, seedings were removed and total RNA was extracted using EasyPure Plant RNA Kit (ER301-01; TransGen Biotech, Beijing, China) following manufacturer instructions. Integrity of RNA samples was checked on 1% Agarose gel. RNA quantification was performed in a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and samples with A260/A280 in a range of 1.9–2.1 were considered for cDNA synthesis. The cDNA was synthesized using ABClonal ABScript III RT Master Mix supplemented with gDNA remover. The reverse transcription reaction components included a 4 μL 5X ABScript III RT Mix, 1 μL 20X gDNA remover mix, 1 μg total RNA, and 13 μL nuclease-free water making a final volume up to 20 μL. The reaction conditions for the reaction were 37 °C for 2 minutes, 55 °C for 15 minutes, 85 °C for 5 minutes, and 4 °C to hold. The products were quantified using Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and stored at −20 °C for subsequent qRT-PCR reaction.
6
Genomic DNA and RNA Extraction from Pig Tissues
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Genomic DNA was extracted from ear tissues of pigs by phenol/chloroform and ethanol precipitation [8 ]. The quality and quantity of all DNA samples were assessed by 1% agarose gel electrophoresis and NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA), respectively. Total RNA from the blood was isolated by a TRI Pure LS Reagent (Beijing BioTeke Corporation, Beijing, China). The total RNA from other tissues was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and quantified as indicated for DNA using the NanoDrop 2000 Spectrophotometer (Thermo Scientific, USA). The integrity of the RNA samples were detected by 1% agarose gel electrophoresis before the first-strand cDNA was synthesized. RNA was purified and reversely transcribed into cDNA using the PrimerScript RT Reagent Kit with gDNA Eraser (TaKaRa Biotechnology Co., Ltd, Dalian, China) following the manufacturer’s instructions.
7
RNA Extraction and cDNA Synthesis
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Total RNA was isolated with TRIzol reagent (Invitrogen) and concentration and quality were assessed with Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize cDNA of 1 μg RNA, following manufacturer’s recommendations.
Genomic DNA was obtained from undifferentiated BMSCs in the third passage, using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions. DNA concentration was assessed with Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and quality was analyzed by electrophoresis on 2 % agarose gel. For PCR reactions, DNA was diluted to 100 ηg/μL.
Genomic DNA was obtained from undifferentiated BMSCs in the third passage, using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions. DNA concentration was assessed with Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and quality was analyzed by electrophoresis on 2 % agarose gel. For PCR reactions, DNA was diluted to 100 ηg/μL.
8
Nucleic Acid Extraction from Frozen Tissues
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Frozen mouse tissues were digested in lysis buffer (35 mM EDTA, 75 mM NaCl, 10 mM Tris-HCl pH 8.0, 1% SDS, and 2 mg ml–1 Proteinase K) overnight and extracted using the phenol : chloroform method. Genomic DNA quality and quantity were examined on a 1% agarose gel, a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific), and a Qubit 2.0 fluorometer (Invitrogen).
For RNA studies, frozen tissues were treated with RNAlater-ICE Frozen Tissue Transition Solution (Ambion) and total RNA was extracted with an RNeasy Mini Kit (Qiagen). Total RNA was quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Kit (Agilent). The investigated RNA samples had minimum RNA Integrity Number of 7.5.
For RNA studies, frozen tissues were treated with RNAlater-ICE Frozen Tissue Transition Solution (Ambion) and total RNA was extracted with an RNeasy Mini Kit (Qiagen). Total RNA was quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Kit (Agilent). The investigated RNA samples had minimum RNA Integrity Number of 7.5.
9
Microbial Community Analysis of Neonatal Fecal and Maternal Milk Samples
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Microbial DNA was extracted from the neonatal fecal samples using a QIAamp DNA Stool Mini Kit (Qiagen, Duesseldorf, Germany). Microbial DNA was extracted from the maternal milk using a DNeasy PowerFood Microbial Kit (Qiagen). Both procedures were carried out according to the manufacturer’s instructions, with minor modifications. The extracted DNA was quantified using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, DE, USA), and the DNA integrity was determined by 1% agarose gel electrophoresis. Amplification of the V4 region of bacterial 16S r RNA genes was carried out as previously described [26 (link)]. In brief, barcoded universal primers 515F and 806R were designed for PCR amplification with initial denaturation at 95 °C for 5 min and 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 45 s, followed by a final extension at 72 °C for 10 min. The PCR products were gel-purified, quantified via a NanoDrop™ 2000 spectrophotometer (Thermo Scientific), pooled at equal molar ratios, and sequenced on an Illumina HiSeq 2500 platform.
10
Microbial DNA Extraction from Diverse Samples
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Microbial DNA was extracted from the intestinal mucosa-associated microbiota, feces, and swabs using QIAamp DNA Stool Mini Kit (Qiagen, Duesseldorf, Germany). Microbial DNA was extracted from the milk using a DNeasy PowerFood Microbial Kit (Qiagen). Both procedures were carried out according to the manufacturer’s instructions, with an addition of a bead-beating step using 0.25 g of 0.15 mm garnet beads and 0.25 g of 0.1 mm zirconia beads. DNA was quantified with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, DE, USA), and the integrity was checked by 1% agarose gel electrophoresis. Amplification of the V3-V4 region of bacterial 16S rRNA genes was carried out as previously described [84 (link)]. Briefly, bar-coded universal primers 341F and 806R were designed for PCR amplification with initial denaturation at 95 °C for 5 min and 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 45 s, followed by a final extension at 72 °C for 10 min. The PCR products were gel purified, quantified via NanoDrop™ 2000 spectrophotometer (Thermo Scientific), pooled at equal molar ratios, and sequenced on Illumina HiSeq 2500.
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