Total RNA was extracted from half a rectal mucosal biopsy using the miRNeasy Mini Kit (Qiagen, UK) following the manufacturer's instructions. Tissue disruption was performed by shaking the tissues with five 3 mm glass beads (VWR, UK) for 1 m in QIAzol Lysis Reagent (miRNeasy Mini Kit) using an amalgamator. The lysate and beads were transferred to QiaShredders (Qiagen, UK) for homogenization by centrifugation for 2 m at maximum speed. RNA was eluted in 30 µL of RNase‐free water. Concentration and RNA purity were determined using the NanoDrop 1000 spectrophotometer (Thermo Scientific) and NanoDrop 1000 Software version 3.7.1. RNA integrity was assessed by agarose gel electrophoresis.
Nanodrop 1000
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, China, Japan, Australia, Canada, Italy, France, Switzerland, Ireland, Denmark, Belgium, Norway, Spain, Portugal, Jamaica, Austria, Lithuania, Singapore
The NanoDrop 1000 is a spectrophotometer designed for the quantification and analysis of small volume samples. It can measure the absorbance of samples ranging from 1 to 2 microliters in volume. The device uses innovative sample-retention technology to allow for direct measurement without the need for cuvettes or other sample vessels.
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Rneasy plus mini kit, by Qiagen (92 mentions)
Tri reagent, by Merck Group (91 mentions)
Dnase 1, by Thermo Fisher Scientific (90 mentions)
Iscript cdna synthesis kit, by Bio-Rad (83 mentions)
Dneasy blood and tissue kit, by Qiagen (78 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (74 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (74 mentions)
Quantitect reverse transcription kit, by Qiagen (68 mentions)
Primescript rt reagent kit, by Takara Bio (67 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (65 mentions)
Rneasy kit, by Qiagen (65 mentions)
Lightcycler 480, by Roche (59 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (59 mentions)
2 999 protocols using nanodrop 1000
1
Total RNA Extraction from Rectal Biopsy
2
Solubility Determination of 1G244
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The solubility dependence of 1G244 on DMSO concentration was determined by measuring its absorbance peak at 262 nm relative to a 100% DMSO dilution standard curve (NanoDrop 1000, Thermo Fisher). 1G244 samples were diluted in water and incubated at 40°C for 24 h at 300 r min−1 shaking. Then, samples were spun down for 15 min at 20 000 g. Afterwards, samples were filtrated using a polyvinylidene fluoride filter of 0.22 µm (Millex-GV). The absorbance of the transparent eluted solution was measured at 25°C (S. Dalziel & K. Phizackerley, APPLICATION NOTE: NanoDrop 1000, Small Molecule Crystallography, Thermo Fisher Scientific, Wilmington, Delaware, USA). The absorbance of 1G244 at 25% DMSO was just above the assay noise signal. Thus, estimation of the saturation concentration at lower DMSO% was obtained by interpolation.
3
Quantitative RT-PCR Analysis of Tissue RNA
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Total ribonucleic acid (RNA) was extracted from 25 mg of frozen liver and brain tissues using the PureLink RNA Mini Kit (Life Technologies, Burlington, Canada) and was measured using a NanoDrop-1000 (Thermo Fisher Scientific, Burlington, Canada). Reverse transcription was performed with 1 μg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad). cDNA products were quantified using a NanoDrop-1000 (Thermo Fisher Scientific). qRT-PCR was performed in 20 μl reaction volumes using 50 ng of complimentary DNA (cDNA). Primer pairs were synthesized by the University of Calgary Core DNA Services (Table S1 ). The PCR conditions were as follows: 95°C—2 min, 40 cycles of (95°C—30 s, 60°C—30 s, 72°C—30 s), 72°C—2 min. Samples were run in triplicate on the same reaction plate using the CFX96 Real-Time PCR Detection System (Bio-Rad) with β-actin as a loading control. Data analysis was performed in accordance with previously published work using the 2−ΔΔCt method (Lee et al., 2012 (link)).
4
RNA Isolation from Cells, Exosomes, and FFPE Tissue
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TotalRNA was isolated from cells and exosomes using miRNeasy Mini Kit (Qiagen; Germany). Previously, free-circulating RNA was removed by resuspending the exosome pellet in 100 μl PBS and treatment with 10 units RNase ONE™ Ribonuclease (Promega; United States) for 30 min at room temperature. RNase reaction was stopped by adding 10 units RiboLockRNAse Inhibitor (Thermo Fisher Scientific; United States) to the mixture and incubation for 10 min at room temperature. Afterwards the isolation was performed according to a modified manufacture protocol. RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific; United States). MiRNA of urine exosomes was isolated according to the protocol of cells and in vitro exosomes.
TotalRNA of formalin-fixed paraffin-embedded (FFPE) primary bladder tumors was isolated using miRNeasy FFPE Kit (Qiagen, Germany). 20 sections of each tumor were prepared on a microscope slides to perform a macro-dissection of tumor areas. Tumor sections were transferred into a reaction tube. Afterwards, isolation of totalRNA was performed according to the manufacture protocol and RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific; United States).
TotalRNA of formalin-fixed paraffin-embedded (FFPE) primary bladder tumors was isolated using miRNeasy FFPE Kit (Qiagen, Germany). 20 sections of each tumor were prepared on a microscope slides to perform a macro-dissection of tumor areas. Tumor sections were transferred into a reaction tube. Afterwards, isolation of totalRNA was performed according to the manufacture protocol and RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific; United States).
5
Liver RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from 25 mg of frozen liver tissue using the PureLink RNA Mini Kit (Life Technologies) and was measured using a NanoDrop-1000 (Thermo Fisher Scientific). Reverse transcription was performed with 1 μg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad). cDNA products were quantified using a NanoDrop-1000 (Thermo Fisher Scientific). qRT-PCR was performed in 20 μl reaction volumes using 50 ng of cDNA. Representative genes, 3 upregulated and 3 downregulated, were randomly selected for measurement using qRT-PCR to verify DNA microarray results. qRT-PCR primers were synthesized by the University of Calgary Core DNA Services (Table 1 ). The qRT-PCR conditions were as follows: 95°C−2 min, 40 cycles of (95°C−30 s, 60°C−30 s, 72°C−30 s), 72°C−2 min. Samples were run in triplicate on the same reaction plate using the CFX96 Real-Time PCR Detection System (Bio-Rad) with β-actin as a loading control. Data analysis was performed in accordance with previously published work using the 2−ΔΔCt method (Lee et al., 2012 (link)).
6
RNA Isolation and cDNA Synthesis Workflow
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Total RNA was prepared by the method of Sánchez et al. [56 (link)]. The concentration and quality of total RNA were determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and checked on 1% agarose gels before proceeding (see Additional file 4 ). Approximately 1 μg total RNA was converted into cDNA using a SMART cDNA synthesis kit (Clontech, Palo Alto, CA, USA), optimizing the conditions to obtain a large quantity of clean cDNA in a small volume. For second-strand synthesis, PCR was carried out using a small aliquot (1/10th volume) of the primary template and Advantage 2 Polymerase Mix (Clontech). The thermal cycling program was as follows: initial denaturation at 95°C for 60 s, followed by 18 cycles of denaturation at 95°C for 15 s and annealing at 65°C for 30 s, followed by extension at 68°C for 3 min. For each sample and all PCR reactions, the PCR products were purified using a PureLink PCRTM purification kit (Invitrogen, Carlsbad, CA, USA). Double-stranded cDNA was quantified with a spectrophotometer (NanoDrop 1000, Thermo Scientific). The products were checked on a 2% agarose gel to verify cDNA quality and fragment length.
7
Plasma RNA Extraction and Purification
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Total RNA was extracted and purified using miRNeasy ® Mini kit (Qiagen, German), following the manufacturer's instructions. RNA concentrations were determined with a NanoDrop 1000 (Thermo Scientific, Wilmington, USA). In general, RNA concentrations 8-15 ng/μL, and 100 ng of total RNA per sample were used in the subsequent miRNA microarray.
Plasma sample of 300 μL was mixed with TRIzol ® LS reagent (Invitrogen, USA) with a ratio of 1:3 in 1.5 mL microcentrifuge tube, and incu-bated at room temperature for 5 min. Then, 250 μL chloroform was added and mixed vigorously by vortex. Incubated at room temperature for 15 min and centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant (500 μL per tube) was transferred to an Eppendorf tube. Subsequently, the RNA was precipitated by adding 250 μL isopropanol to the aqueous phase. After being incubated at room temperature for 10 min and centrifuged at 12,000 rpm for 10 min at 4°C, the RNA pellet was rinsed, airdried, and then resuspended in 23 μL RNAase-free water. RNA concentration was determined with a NanoDrop 1000 (Thermo Scientific, Wilmington, USA). This RNA was used in the subsequent RT--qPCR. The low RNA concentration precluded the reliable assessment of RNA purity by A260/A280 and A260/A230 absorbance ratios, and it was also too low for assessment of RNA integrity by Agilent Bioanalyzer "RIN" number.
Plasma sample of 300 μL was mixed with TRIzol ® LS reagent (Invitrogen, USA) with a ratio of 1:3 in 1.5 mL microcentrifuge tube, and incu-bated at room temperature for 5 min. Then, 250 μL chloroform was added and mixed vigorously by vortex. Incubated at room temperature for 15 min and centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant (500 μL per tube) was transferred to an Eppendorf tube. Subsequently, the RNA was precipitated by adding 250 μL isopropanol to the aqueous phase. After being incubated at room temperature for 10 min and centrifuged at 12,000 rpm for 10 min at 4°C, the RNA pellet was rinsed, airdried, and then resuspended in 23 μL RNAase-free water. RNA concentration was determined with a NanoDrop 1000 (Thermo Scientific, Wilmington, USA). This RNA was used in the subsequent RT--qPCR. The low RNA concentration precluded the reliable assessment of RNA purity by A260/A280 and A260/A230 absorbance ratios, and it was also too low for assessment of RNA integrity by Agilent Bioanalyzer "RIN" number.
8
DNA Extraction from Blood Samples
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Genetic material was extracted from peripheral blood collected in EDTA tubes and using an Axy PrepTM Blood Genomic DNA Miniprep kit (Axygen Biotechnology, San Francisco, CA, USA), following the manufacturer’s instructions. The DNA concentration and purity were measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific NanoDrop 1000, NanoDrop Technologies, Wilmington, DE, USA).
9
High-Quality Genomic DNA Extraction
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Cells from the trio cell line were washed with PBS, resuspended in cell suspension buffer, and embedded in thin low-melting-point agarose layers (CHEF Genomic DNA Plug Kit, Bio-Rad). The thin agarose layers were incubated with lysis buffer and proteinase K for 4 hr at 50°. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to 4 hr of drop-dialysis. It was quantified using Nanodrop 1000 (Thermal Fisher Scientific) and/or a Quant-iTdsDNA Assay Kit (Invitrogen/Molecular Probes), and the quality was assessed using pulsed-field gel electrophoresis.
10
High-molecular weight DNA Extraction
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High-molecular weight (HMW) DNA extraction was performed as recommended for the CHEF Mammalian Genomic DNA Plug Kit (BioRad #170-3591). Briefly, cells from the YH or NA12878 cell lines were washed with 2x with PBS and resuspended in cell resuspension buffer, after which 7.5 × 105 cells were embedded in each gel plug. Plugs were incubated with lysis buffer and proteinase K for four hours at 50°C. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to four hours of drop dialysis (Millipore, #VCWP04700) and quantified using Nanodrop 1000 (Thermal Fisher Scientific) and/or the Quant-iT dsDNA Assay Kit (Invitrogen/Molecular Probes).
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