Total RNA was isolated from 20 samples (collected from Henan Provincial People’s Hospital), including ten tumor tissues and ten adjacent normal tissues, using an mirVana™ RNA isolation kit (AM1561, Thermo Fisher, USA). RNA clean-up was performed using RNasey Mini Kit (Qiagen p/n 74,104). Quantification and quality control were achieved by NanoDrop ND-2100 (ThermoFisher) and Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA). cDNA synthesis and biotin labeling of cRNA were performed using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, USA) and the GeneChip™ Hybridization, Wash and Stain Kit (Affymetrix) following the manufacturer’s protocol. The purified and fragmented labeled cRNAs were hybridized onto the Affymetrix PrimeView™ Human Gene Expression microarray. After washing and staining, the arrays were scanned and analyzed using Affymetrix Scanner 3000 (Affymetrix). GeneChip Command Console Software (version 4.0, Affymetrix) and Genespring software (version 14.9, Agilent Technologies) were used to analyze the transcriptome data. Differentially expressed genes (DEGs) from TCGA were analyzed using DESeq2 package within R language (version 3.4.3).
Nanodrop nd 2100
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop ND-2100 is a spectrophotometer designed for the measurement of small sample volumes. It is capable of measuring the absorbance of samples between 220 and 750 nanometers wavelength. The instrument uses a patented sample retention system that requires only 1-2 microliters of sample to perform measurements.
Automatically generated - may contain errors
Lab products found in correlation
Affymetrix scanner 3000, by Thermo Fisher Scientific (7 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Genespring software, by Agilent Technologies (4 mentions)
Agilent 2100, by Agilent Technologies (4 mentions)
Mirvana rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Genechip command console software, by Thermo Fisher Scientific (2 mentions)
Rnasey mini kit, by Qiagen (2 mentions)
One cycle target labeling and control reagents, by Thermo Fisher Scientific (2 mentions)
Affymetrix genechip command console software, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Steponeplus instrument, by Thermo Fisher Scientific (1 mentions)
Fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Expression console, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Mirvanatm rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Affymetrix genechip command console, by Thermo Fisher Scientific (1 mentions)
Trizol, by Merck Group (1 mentions)
10 protocols using nanodrop nd 2100
1
Transcriptome Profiling of Tumor and Normal Tissues
2
Total RNA Extraction and Microarray Analysis
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Total RNA from C666-1 and C666-1R cells, extracted using the TRIzol reagent, was quantified using the NanoDrop ND-2100 (Thermo Scientific, USA), and the RNA integrity was assessed using Agilent 2100 (Agilent Technologies, USA). The sample labeling, microarray hybridization, and washing was performed according to the manufacturer’s instructions. Briefly, total RNA was tailed with poly A and labeled with biotin, after which the labeled RNA was hybridized onto the microarray. After washing, the slides were stained and the arrays were scanned by the Affymetrix Scanner 3000 (Affymetrix, USA).
3
Kidney miRNA Expression Profiling
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Total RNA isolated from kidney samples of db/m, db/db, and db/db + Res mice was quantified with a NanoDrop ND-2100 (Thermo Scientific, Thermo Fisher Scientific, MA, USA) and RNA integrity was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies). Sample labeling, microarray hybridization, and washing were performed according to the manufacturer’s standard protocols. Briefly, total RNA was tailed with poly-A and then labeled with biotin. The labeled RNAs were hybridized onto the Affymetrix miRNA 4.0 microarray. After washing and staining of the slides, the arrays were scanned with an Affymetrix Scanner 3000 (Affymetrix). Affymetrix GeneChip Command Console software (version 4.0, Affymetrix) was used to analyze array images to get raw data and for RMA normalization. GeneSpring software (version 12.5; Agilent Technologies) was used for data analysis. Differentially expressed miRNAs were identified on the basis of a fold-change ≥ 2.0.
4
Isolation and Validation of Small RNA from Rat Lungs
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Rat lungs were homogenized and total RNA (enriched for small RNA) was extracted according to manufacturer's instructions using the mirVana miRNA isolation kit (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and RNA concentration was quantified using NanoDrop ND-2100 (Thermo Fisher Scientific, Inc.). The RNA integrity was assessed using Agilent 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's instructions. To ensure a robust analysis, total RNA was assessed by electrophoresis and the RNA integrity number (RIN). Only samples with acceptable XVIIIS and 28S peaks and RIN values greater than eight were included for miRNA profile analysis.
5
Serum miRNA Quantification and Normalization
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Total RNA was isolated from the serum using the total RNA purification kit. Briefly, RNA was isolated from 150 μl of serum using the RL buffer washed and eluted in RNAse free water. RNA concentration and purity were quantified using NanoDrop ND-2100 (Thermo Fisher Scientific, USA). To normalize between samples, 3.5 μl Caenorhabditis elegans miR-39 (cel-miR-39) was added to each sample. Immediately after RNA isolation, 5 μl of RNA was reverse transcribed using the microScript microRNA cDNA synthesis kit. cDNA was PCR-amplified (StepOnePlus instrument, Applied Biosystems, USA) using RealQ Plus Master Mix Green, high ROX and miRNA-specific primers for miR-126 and miR-27a. All samples were assayed in duplicate. Relative expression level for a given miRNA was normalized to cel-miR-39 as external control. The expression was calculated as fold change according to the formula: fold change = 2–ΔΔCT in which ΔΔCT = [(CT gene − CT cel − miR − 39)treatment–[CT gene − CT cel − miR − 39)]CTL [33 (link)]. The forward primer sequences of miRs were as follows: miR-126: 5′-TCGTACCGTGAGUTATAATGCG-3′, miR-27a: 5′-TTCACAGTGGCTAAGTTCCGC-3′, and cel-miR-39: 5′-UCACCGGGUGUAAAUCAGCUUG-3′.
We used a universal primer that was supplied by the company as reverse in the reactions.
We used a universal primer that was supplied by the company as reverse in the reactions.
6
Profiling Liver miRNA Expression
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Total RNAs were isolated from liver tissues and quantified by the NanoDrop ND-2100 (Thermo Scientific). After the control of RNA integrity using Agilent 2100 (Agilent Technologies), total RNAs were tailed with Poly A, labeled with Biotin, and then hybridized for 16 hrs at 48°C on Affemetrix miRNA 3.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. The arrays were scanned by the Affymetrix Scanner 3000 (Affymetrix) and the array images were analyzed using Affymetrix GeneChip Command Console 4.0 software (Affymetrix) to get raw data and then provide RMA normalization. Using Genespring 12.5 software (Agilent Technologies), the probes that at least 75% of samples in any 1 condition out of 2 conditions have flags in “P” were chosen for further data analysis. The differentially expressed miRNAs, with a fold change>= 2.0 and a P value < 0.05 between the groups PH-0h and PH-48h, were chosen for further validation using qRT-PCRs. The MIAME-compliant data have been submitted to Gene Expression Omnibus (GEO, platform ID: GSE68451).
7
Cardiac miRNA Profiling Protocol
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Total RNAs were isolated from heart tissues using mirVana™ RNA Isolation Kit, quantified by NanoDrop ND-2100 (Thermo Scientific), and controlled for RNA integrity using Agilent Bioanalyzer 2100 (Agilent Technologies) according to the manufacturer's instructions. miRNA profiling was performed with OE Biotech's (Shanghai, China) miRNA microarray service. The arrays from the control group are the same as we previously used [14 (link)].
8
RNA Extraction and Microarray Analysis
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Total RNA was extracted using the TRIzol reagent (15596018; Invitrogen, New York, USA) following the manufacturer’s protocols. The quality of the purified RNA was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, California, USA). Total RNA was quantified using NanoDrop Nd-2100 (Thermo Fisher Scientific, Massachusetts, USA), and RNA integrity was detected using an Agilent 2100 bioanalyzer (Agilent Technologies). After passing the quality inspection, the samples were labeled, hybridized, and eluted according to the chip’s standard process. The original images were then scanned using an Affymetrix Scanner 3000 (Affymetrix, USA), and the images were analyzed using Expression Console (version 1.3.1, Affymetrix) data analysis software, converting the resulting raw data into range migration algorithm (RMA) normalized algorithm signal values. The expression patterns of RNAs (microRNA/miRNA/miR) among samples were analyzed by hierarchical cluster analysis. Meanwhile, we used quantitative reverse transcription-PCR (qRT‑PCR) to verify the expression of key miRNAs in different experimental groups. miRNAs exert their regulatory function by limiting the expression of their target. Hence, we queried the target genes of miR-221-3p using the TargetScan database (http://www.targetscan.org/vert ) and found that P27 and miR-221-3p had good complementarity in both humans and rats.
9
Transcriptome Analysis of Tumor and Normal Tissues
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Total RNA was isolated from 20 samples, including ten tumor tissues and ten adjacent normal tissues, using an mirVana TM RNA isolation kit (AM1561, Thermo Fisher, USA). RNA clean-up was performed using RNasey Mini Kit (Qiagen p/n 74104). Quanti cation and quality control were achieved by NanoDrop ND-2100 (ThermoFisher) and Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA). cDNA synthesis and biotin labeling of cRNA were performed using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, USA) and the GeneChip TM Hybridization, Wash and Stain Kit (Affymetrix) following the manufacturer's protocol. The puri ed and fragmented labeled cRNAs were hybridized onto the Affymetrix PrimeView TM Human Gene Expression microarray. After washing and staining, the arrays were scanned and analyzed using Affymetrix Scanner 3000 (Affymetrix). GeneChip Command Console Software (version 4.0, Affymetrix) and Genespring software (version 14.9, Agilent Technologies) was used to analyze the transcriptome data. Differentially expressed genes (DEGs) from The Cancer Genome Atlas (TCGA) were analyzed using DESeq2 package within R language (version 3.4.3).
10
Mandibular Gene Expression Analysis
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The mandible was prepared from sham-operated mice and OVX mice. The left mandible from the sham-operated group and OVX group were harvested using TRIzol (Sigma-Aldrich) to extract the total RNA. The total RNA was quantified with NanoDrop ND-2100 (Thermo Scientific), and RNA integrity was assessed using Agilent 2100 (Agilent Technologies). Sample labeling, microarray hybridization, and washing were performed based on the manufacturer's standard protocols. Briefly, the total RNA was tailed with Poly A and then labeled with Biotin. Afterward, the labeled RNAs were hybridized on the microarray. After washing and staining the slides, the arrays were scanned with Affymetrix Scanner 3000 (Affymetrix). The software Affymetrix GeneChip Command Console (version4.0, Affymetrix) was used to analyze the array images to obtain raw data and then conduct RMA normalization. Next, Genespring software (version 12.5; Agilent Technologies) was used to conduct the subsequent data analysis.
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