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Denaturing solution

Manufactured by Thermo Fisher Scientific
Sourced in United States

Denaturing solution is a chemical reagent used in various laboratory protocols to disrupt the secondary and tertiary structures of proteins, converting them to their primary structure. This solution can be used in techniques such as gel electrophoresis, Western blotting, and protein purification to facilitate the analysis and separation of proteins.

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26 protocols using denaturing solution

1

Whole Blood RNA Extraction and Preservation

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Peripheral blood was collected at the time points foreseen by the study design in EDTA tubes with 400 μL of 2× Denaturing solution (Ambion, Austin, TX, USA) and stored at − 20 °C. Total RNA was extracted from 800 μL of blood with the MirVana Paris Kit and treated with Turbo DNA-free Kit (Ambion). RNA concentration was estimated with a Nanoquant Infinite M200 instrument (Tecan, Austria). RNA quality was assessed on an Agilent Bioanalyzer using the RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA), and samples with RNA integrity number > 7.5 were considered acceptable for processing.
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2

Plasma miRNA Isolation and Quantification

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Total RNA was isolated from 400 μL of plasma using the mirVanaTM PARISTM Kit (Ambion, Austin, TX) according to the manufacturer’s instructions with modification. For normalization of sample-to-sample variation, 25 fmol of synthetic C.elegans miRNA cel-miR-39 (Qiagen, Germany) was added to each sample after addition of 2 × Denaturing Solution (Ambion, Austin, TX) [24]. RNA was dissolved in 100 μL of RNase-free water, and then stored at −80 °C until analysis. Total RNA was reverse transcribed using the DRR037A PrimeScript® RT Master mix (Takara, Dalian, P.R. China). The RT reaction was incubated at 37 °C for 15 min, at 85 °C for 5 s, and then held at 4 °C. The cDNA product was stored at −20 °C until analysis.
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3

Extraction and Quantification of Serum/Exosomal miRNA

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The mirVana PARIS Kit (Ambion, Austin, TX) was used to extract total RNA from 200 µL serum or exosomes following the given protocol. For normalization, 5 µL of synthetic C. elegansmiR‐39 (5 nM/L, RiboBio, Guangzhou, China) was added to each sample after the addition of denaturing solution (Ambion, Austin, TX). Tissue samples were processed using Trizol (Invitrogen, Carlsbad, CA) to extract total RNA. Acquired total RNA was lysed in 100 µL RNase‐free water and restored at −80°C until use. The NanoDrop ND‐1000 spectrophotometer (NanoDrop, Wilmington, DE) was used to measure the concentration and purity of RNA.
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4

Gastric Cancer Tissue and Plasma RNA Extraction

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All the GC specimens including tissues and blood, were obtained from patients who received surgical resection for GC at Shanghai General Hospital affiliated of Shanghai Jiaotong University. Before RNA extraction, all specimens were snap-frozen instantly and stored at −80C. Blood samples (5 mL) were collected from all subjects in EDTA tubes and centrifuged at 3,000 rpm for 10 min at 4C, then the plasma was cautiously collected and also keep it at −80C until use. Total RNA extraction from tissues and plasma samples used TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and TRNzol A+ (TIANGEN, Beijing, China). miRNA used miRcute Serum/plasma miRNA isolation kit (TIANGEN, Beijing, China) according to the manufacturer’s protocols. After adding denaturing solution (Ambion) for normalization of the sample-to-sample variation, 1 ul of synthetic external control (1 umol/L; TIANGENN) was spiked into each sample.
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5

Total RNA Extraction from Biofluids and Tissues

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For plasma and exosome samples, total RNA was isolated using the mirVana PARIS Kit (Ambion) following the manufacturer's protocol. After the addition of denaturing solution (Ambion), 5 μL synthetic C elegans miRNA cel‐miR‐39 (5 nM/L, RiboBio) was added to each sample to normalize sample‐to‐sample variation. For tissue specimens, we used TRIzol (Invitrogen) to extract total RNA in accordance with the manufacturer's instruction. The acquired total RNA was finally eluted into 100 μL of RNase‐free water and kept at −80°C until further use. The concentration and purity of total RNA was measured using a Nanodrop 2000 spectrophotometer (NanoDrop Technologies). Samples with total RNA concentration <10 ng/μL were excluded in the analysis.
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6

Serum and Exosome RNA Extraction

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Total RNA was extracted from 200μl serum or exosome using the mirVana PARIS Kit (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. 5μl of synthetic C.elegans miR-39 (5 nM/L, RiboBio, Guangzhou, China) was added to each sample after the addition of denaturing solution (Ambion, Austin, TX, USA) for normalization. Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from tissue samples. Finally, total RNA was dissolved in 100μl RNase-free water and kept at -80°C until further analysis. The ultraviolet spectrophotometer was applied to evaluate the concentration and purity of the extracted total RNA.
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7

Exosomal miRNA Profiling by qRT-PCR

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Total RNAs (including miRNAs) derived from exosomes were extracted by a MirVana Paris Kit (Ambion, Austin, TX, USA) according to the manual. Denaturing solution (Ambion, Austin, TX, USA) was added to each sample. Total RNAs were dissolved in 100 μL RNase-free water. For further measurement, samples were stored in a −80°C refrigerator. The concentration of RNAs was assessed by a Nanodrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). After that, reverse transcription was applied to synthesize cDNA using the TaqMan Micro-RNA Reverse Transcription Kit (Thermo Fisher Scientific). The expression of ex-miRNAs was quantified by qRT-qPCR analysis according to the instructions of the manufacturer. We examined the obtained cDNA by qRT-PCR with TaqMan microRNA primers specific for miR-31, miR-192, and miR-375 (Thermo Fisher Scientific Inc., Waltham, MA, USA). A 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA) was employed for amplification and evaluation for miRNAs extracted from the exosomes. The miRNA expressions were calculated by 2−ΔΔCt relative to RNU6B (U6): ΔCt = CtmiRNA − CtU6.
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8

Isolation of Total RNA from Plasma and Tissues

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Total RNA from 200 μl plasma or exosomes was isolated using the mirVana PARIS Kit (Ambion, Austin, TX, USA) in accordance with the protocol. After the addition of denaturing solution (Ambion, Austin, TX, USA), 5 μl synthetic C.elegans miR-39 (5 nM/L, RiboBio, Guangzhou, China) was spiked into each sample for normalization of variation between samples. According to the manufacturer's instructions, Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from tissue samples. Total RNA was lysed in 100 μl RNase-free water and stored at −80°C for further analysis. The concentration and purity of the total RNA was analyzed using the ultraviolet spectrophotometer.
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9

Total RNA Extraction from Serum and Tissue

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Total RNA was extracted from 200 μL serum or exosomes using the mirVana PARIS Kit (Ambion, Austin, TX, USA) following the given protocol. 5 μL of synthetic C.elegans miR-39 (5 mmol/L, RiboBio, Guangzhou, China) was added for sample-to-sample normalization after the addition of denaturing solution (Ambion, Austin, TX, USA). Total RNA of tissue samples was obtained using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. After being extracted, total RNA was dissolved in 100 μL RNase-free water and stored at −80°C until analysis. The concentration and purification of the RNA sample were assessed by the ultraviolet spectrophotometer.
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10

Plasma RNA Extraction with Normalization

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The total RNA from 400 μl plasma was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using an RNeasy Mini kit (Qiagen, Hilden, German) according to the manufacturer’s instructions. Briefly, 10 nM synthetic C. elegans miR-39 (RiboBio, China) was spiked-in to each sample after the addition of denaturing solution (Ambion, Austin, TX, USA) for normalization of the between-sample variation. RNA concentrations were determined by measuring the sample absorbance at 260 nm with the ultraviolet spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
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