The HFD mice with or without 400 mg/kg/BW/day of LMF-HSFx through oral gavage. After 16 weeks treatment, the mice were anesthetized by inhalant anesthesia with isoflurane before sacrifice. RNA was extracted from adipose tissues by using a Direct-zol RNA Miniprep Plus kit (Cat. No. R2073, Zymo Research, Irvine, CA, USA) and its quantity and quality were estimated by NanoDrop measurement (ThermoFisher, Waltham, MA, USA). The RT PCR processing was performed using a commercially available RT2 Profiler™ PCR Array Mouse Adipogenesis (Cat. no. PAMM-049ZA, QIAGEN, Hilden, Germany) for genes of interest. Following, the fold change of target gene shows the ratio of treated group/control group and values > 1.5 or < 0.5 were considered significantly modified.
Nanodrop measurement
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop measurement is a spectrophotometric instrument designed for the quantification and analysis of nucleic acids and proteins. It utilizes a small sample volume, typically 1-2 microliters, to measure the absorbance of the sample and determine its concentration.
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Lab products found in correlation
Truseq rapid sbs kit, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Truseq rapid pe cluster kit, by Illumina (2 mentions)
2 mercaptoethanol, by Bio-Rad (2 mentions)
Lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Cdp star substrate, by Thermo Fisher Scientific (2 mentions)
I block, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rt2 profiler pcr array mouse adipogenesis, by Qiagen (1 mentions)
Dna extraction kit, by Bioneer (1 mentions)
Superscript vilo cdna synthesis kit, by Roche (1 mentions)
Dreamtaq green pcr mix, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex, by Thermo Fisher Scientific (1 mentions)
Rt2 profiler pcr array, by Qiagen (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
16 protocols using nanodrop measurement
1
Adipogenesis Gene Expression in HFD Mice
2
Blood and DNA Analysis of Iranian Lori-Bakhtiari Ewes
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One hundred blood samples were collected from six‐month‐old indigenous Iranian fat‐tailed ewes, belonged to the Lori‐Bakhtiari breed, with normal delivery and similar grazing history. Lori‐Bakhtiari breed is originated from Chaharmahal and Bakhtiari province in the west of Iran and kept mainly for meat production. This population was maintained under semi‐extensive farm conditions, with no selection or immigration programs. Vaccination was carried out on the flock as used locally: Brucellosis vaccine (strain Rev.1, Razi, Iran), Anthrax vaccine (strain 34F2, Razi, Iran) and Sheep Pox vaccine (strain RM/65, Razi, Iran) at the age of three months; Ovine and Caprine Clostridial vaccine (polivac®, Vetal, Turkey) at week 8 and repeated 21 days later; Agalactica (Mycoplasma agalactiae, Razi, Iran) at the age of three months and repeated two times with a 3‐week interval.
Blood samples were centrifuged to separate the serum and stored at −20°C until further analyses. Genomic DNA was also extracted from the whole blood using commercial DNA extraction kit (Bioneer, Korea) and purity of the DNA was confirmed by NanoDrop measurement (Thermo Scientific, USA) and agar gel electrophoresis. The study procedure was approved by the Animal Care and Use Committee of the Shiraz University. Flock owner's written consent was obtained for sampling and performing this research.
Blood samples were centrifuged to separate the serum and stored at −20°C until further analyses. Genomic DNA was also extracted from the whole blood using commercial DNA extraction kit (Bioneer, Korea) and purity of the DNA was confirmed by NanoDrop measurement (Thermo Scientific, USA) and agar gel electrophoresis. The study procedure was approved by the Animal Care and Use Committee of the Shiraz University. Flock owner's written consent was obtained for sampling and performing this research.
3
Transcriptome Profiling by RNA-Seq
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The quality of the RNA was assessed by Nanodrop measurement (Thermo Fisher Scientific)58 (link). RNA-sequencing (RNA-Seq) libraries were prepared by TruSeq Rapid PE Cluster Kit and TruSeq Rapid SBS kit (Illumina). The libraries were sequenced on Illumina HiSeq. 2500 using a read length of 2 × 150 bp. CASAVA v1.8.2 was conducted and RNA-seq reads were aligned to mouse transcriptome (UCSC gene) and genome (NCBI37/mm9) references respectively using Burrows-Wheeler Aligner. After transcript coordinate was converted to genomic positions, an optimal mapping result was selected either from transcript or genome mapping by comparing the minimal edit distance to the reference. Local realignment was performed within in-house short reads aligner with smaller k-mer size (k = 11). Finally, fragments per kilo base of exon per million fragments mapped (fpkm) values were calculated for each UCSC gene while considering strand-specific information.
4
RNA-Seq Library Preparation and Alignment
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RNA quality was assessed by Nanodrop measurement (Thermo Fisher Scientific). RNA-sequencing (RNA-Seq) libraries were prepared using TruSeq Rapid PE Cluster Kit and TruSeq Rapid SBS kit (Illumina). The libraries were sequenced on Illumina HiSeq 2500 using a read length of 2 × 150 bp. RNA-seq reads were demultiplexed using CASAVA v1.8.2 and aligned to human transcriptome (UCSC gene) and genome (GRCh37/hg19) references respectively using BWA [51 (link)]. After transcripts’ coordinates were converted to genomic positions, an optimal mapping result was selected either from transcript or genome mapping by comparing the minimal edit distance to the reference. Local realignment was performed within in-house short reads aligner with smaller k-mer size (k = 11). Finally, fragments per kilobase of exon per million mapped fragments (fpkm) values were calculated for each UCSC gene while considering strand-specific information. Summary sequencing statistics and data quality metrics are shown in Supplementary Table 17 .
5
Western Blot Analysis of Protein Lysates
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Protein lysates were prepared in a lysis buffer [5 mmol/L MgCl2, 137 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% CHAPS, 10 mmol/L HEPES (pH 7.5)] supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail 2 (both from Sigma, St. Louis, MO), and then normalized using NanoDrop measurement (Thermofisher scientific, Waltham, MA) or Bio-Rad protein assay (Hercules, CA, catalog no. 500–0006). After being denatured in LDS sample buffer (Invitrogen) supplemented with 2-Mercaptoethanol (Bio-Rad, Hercules, CA), protein samples were loaded onto 10% or 14% SDS-PAGE gels which were then transferred electrophoretically onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 0.2% I-Block (Applied Biosystems, Grand Island, NY) in PBS-T (PBS containing 0.1% Tween 20) and incubated with the primary antibodies in PBS-T containing 0.2% I-Block overnight at 4°C or at room temperature for 3 hours. The membranes were then washed and incubated with a secondary antibody in PBS-T containing 0.2% I-Block conjugated to alkaline phosphatase, followed by development with CDP-Star substrate (Applied Biosystems, Grand Island, NY).
6
Quantitative Gene Expression Analysis
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RNA was extracted from cells using an RNeasy Mini kit (Qiagen, Maryland, USA) and its quantity and quality were estimated by NanoDrop measurement (Thermofisher, USA). 500 ng RNA was used for cDNA synthesis (Qiagen) and gene expression monitored by Real-time PCR with SYBR Green master mix using commercially available RT2 Profiler PCR Array and primers for genes of interest (Qiagen, USA). Amplicons were generated on the QuantStudio Flex 12K (Applied Biosystems, California, USA) and gene expression calculated using the comparative threshold cycle method (2−ΔΔCt) after normalising with transcripts encoding HPRT1 and YY1.
For RT-PCR experiments, purified RNA was reverse transcribed into cDNA using SuperScript VILO cDNA synthesis Kit (Roche), according to the manufacturer’s instructions. DNA was amplified by cycles of denaturation (95 °C, 30 s) annealing (primer-specific temperature; 30 s) and extension (72 °C; 30 s) in DreamTaq Green PCR Mix (ThermoFisher) for 35–40 cycles. Final extension was for 5 min at 72 °C. Amplified PCR products were separated and visualised after electrophoresis in 1% agarose gels containing 0.005% GelRed. Vomeronasal receptor primer details are provided in Supplementary Table1 .
For RT-PCR experiments, purified RNA was reverse transcribed into cDNA using SuperScript VILO cDNA synthesis Kit (Roche), according to the manufacturer’s instructions. DNA was amplified by cycles of denaturation (95 °C, 30 s) annealing (primer-specific temperature; 30 s) and extension (72 °C; 30 s) in DreamTaq Green PCR Mix (ThermoFisher) for 35–40 cycles. Final extension was for 5 min at 72 °C. Amplified PCR products were separated and visualised after electrophoresis in 1% agarose gels containing 0.005% GelRed. Vomeronasal receptor primer details are provided in Supplementary Table
7
Comprehensive Biomolecular Extraction Protocol
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DNA was isolated using the QIAamp DNA Mini Kit (Quiagen, Hilden, Germany) according to the manual. RNA from cell lines was isolated using the RNAeasy Mini Kit (Qiagen, Hilden, Germany) according to the manual. Proteins were extracted using RIPA buffer (Cell Signaling, Frankfurt am Main, Germany) containing protease inhibitor tablets (Roche, Basel, Switzerland). Histones were extracted using the Histone Extraction Kit according to the manufacturer protocol (Abcam, Cambridge, UK). Protein concentrations were assessed by the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Schwerte, Germany). RNA concentrations as well as 260/280 nm, 260/230 nm purity ratios were determined by NanoDrop measurement (Thermo Fisher Scientific, Schwerte, Germany).
8
qRT-PCR Quantification of Ebola Virus Genome
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RNA yields were measured with a Nanodrop 1000 (Thermo Scientific) and were normalised to 60ng/μl before addition to qRT-PCR. Primers and minor-groove binder probes were using previously described sequences16 (link) to quantify the relative abundance of the EBOV genome. The reaction was set up with 900 nM of forward and reverse primers, 250 nM probe, 1x TaqMan Fast Virus Mastermix (Life Technologies) to a total volume of 20 μl and tested using the ABi 7500 (Life Technologies) at a thermal sequence of 50 °C for 5 minutes, 95 °C for 20 seconds followed by 40 cycles of 95 °C for 3 seconds and 60 °C for 30 seconds Samples were tested in triplicate and results analysed at a threshold value of 0.2. For gene-specific RT-PCR profiling SYBR Green gene specific PCR assays were obtained as validated commercially available assays (Qiagen). RNA was treated with RNase free DNase (Promega) and cleaned up using the RNEasy MinElute Kit (Qiagen). RNA was equalised between all samples tested using nanodrop measurement (Thermo Scientific). Assays were performed in two stages, with the reverse transcription (RT) step performed with the RT2 First Strand kit (Qiagen) and the gene specific assay performed with gene specific primers (Qiagen) and the RT2 SYBR Green qPCR Mastermix (Qiagen), melt curves were performed to verify specificity of primer sets.
9
Nanoparticle-Mediated siRNA Delivery In Vivo
10
Western Blot Analysis of Protein Lysates
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