Genomic DNA extraction using QuickExtract DNA Extraction Solution (Lucigen) was performed according to manufacturer's instruction. For RNA extraction cells were harvested with 1 ml RNAsolv reagent (Omega Bio-Tek) per 6 well and RNA was isolated according to manufacturer's instruction, with the following changes: instead of 200 μl chloroform, 150 μl 1-Bromo-3-chloropropane (Sigma-Aldrich) was added to the RNAsolv. Additionally, in the last step the RNA pellet was dissolved in 20 μl RNase-free water by incubating for 10 min on a shaking 65 °C heat block.
1 bromo 3 chloropropane
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
1-bromo-3-chloropropane is a halogenated organic compound commonly used as a chemical intermediate in research and industrial applications. It is a colorless, volatile liquid with a density greater than water. The compound contains both bromine and chlorine functional groups, which contribute to its chemical reactivity and versatility.
Automatically generated - may contain errors
Lab products found in correlation
Tri reagent, by Merck Group (22 mentions)
Trizol, by Thermo Fisher Scientific (22 mentions)
Trizol reagent, by Thermo Fisher Scientific (15 mentions)
Rneasy mini kit, by Qiagen (12 mentions)
2 propanol, by Merck Group (8 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (6 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Tri reagent, by Molecular Research Center (5 mentions)
Isopropanol, by Merck Group (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Qiashredder column, by Qiagen (5 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Tissueruptor, by Qiagen (4 mentions)
Qiazol, by Qiagen (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Tissuelyser, by Qiagen (3 mentions)
Trizol, by Merck Group (3 mentions)
85 protocols using 1 bromo 3 chloropropane
1
DNA and RNA Extraction Protocol
2
Retinal mRNA Isolation and Analysis
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Retinas were isolated following trans-cardial perfusion, snap-frozen in liquid nitrogen and stored at −80 °C until use. For mRNA analysis, individual retinas were homogenised in 800 μl TRIzol reagent (ThermoFisher, UK) using motor-driven pellet pestles. After 1 hour incubation, 80 μl of 1-bromo-3-chloropropane (Sigma-Aldrich, UK) was added, the mixture shaken vigorously for 15 seconds and incubated for 3 minutes followed by centrifugation at 12,000 g for 15 minutes at 4 °C. 300 μl of resultant RNA-containing aqueous phase was purified using the PureLink RNA Mini Kit (ThermoFisher, UK) according to the manufacturers’ instructions. 250 ng RNA was transcribed into cDNA using the TaqMan Reverse Transcription Reagents kit (ThermoFisher, UK) following the manufacturers’ instructions for cDNA synthesis with random hexamers. qPCR analysis was performed in duplicate using 1x iTaq Universal SYBR Green Supermix (Bio-Rad, UK) with primers (Supplementary Tables S1 –S3 ). The efficiency of each primer pair was calculated based on five serial dilutions (5-fold) of retinal RNA. Relative RNA levels were calculated using the Pfaffl method49 (link) to take into account differences in primer efficiency compared to the housekeeping gene GAPDH.
3
Optimization of Fetal Bovine Serum Protocols
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Fetal bovine serum (FBS), TaqMan Gene Expression Cells-to-CT Kit (product no. AM1728), electrophoresis markers, Lipofectamine 3000 (product no. L3000001) and GRP78 siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA ). Phosphate-buffered saline (PBS) was purchased from BioWest (Riverside, MO, USA). Medium M199 (product no. M2154), antibiotic–antimycotic solution, Tris, trypsin, FSH (product no. F4021), LH (product no. L5259), IGF1 (product no. I3769), human vaspin (product no. SRP4915), KT5720 (product no. K3761), Laemmli buffer (product no. 38733), Na-deoxycholate, Nonidet NP-40, sodium dodecyl sulfate (SDS), protease inhibitors (ethylene diamine tetraacetic acid-free), dithiothreitol (DTT), Tween 20, bromophenol blue, and 1 bromo-3-chloro-propane were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pig vaspin was not available at the beginning of experiments; therefore, human vaspin was used (there is 90% homology of vaspin nucleotides between these species; sequences were analyzed against the NCBI, USA database using the BLAST program). PD98059 (product no. 1213) was obtained from Tocris (Bristol, GB). Bradford protein assay kit, 4–20% gels (product no. 456-1093) and membranes (product no. 1704156) were obtained from Bio-Rad (Hercules, CA, USA).
4
RNA Extraction from Cell Pellets
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We extracted the total RNA with TRI reagent (Molecular Research Center, Cincinnati, OH, USA). Briefly, 1 mL of TRI reagent was added to the pooled replicates of 3 pellets from 4 independent donors. After homogenization by a Tissue Lyser (Qiagen, Hilden, Germany) for 3 min and 5 Hz, the phase separation by 1-bromo-3-chloropropane (Sigma) in a volume ratio of 1:10 with the TRI reagent was performed. The quantity and quality of the RNA samples were measured by using Nanodrop (Thermo Scientific, Waltham, MA, USA) and Bioanalyzer (Agilent, Santa Clara, CA, USA). RNA sequencing experiments and further data analysis were conducted at QIAGEN Genomic Services (Hilden, Germany).
5
RNA Extraction from Liver and Intestine
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Total RNA was extracted from approximately 100 mg of liver and mid-intestine by homogenisation in 1 ml of TriReagent (Sigma-Aldrich) using a Mini-Beadbeater 24 (Biospec). Phase separation was achieved by the addition of 100 µl of 1-bromo-3-chloropropane (Sigma) and centrifugation at 20 000 g. Subsequently 400 µl of the supernatant were recovered and RNA precipitated by the addition of 200 µl isopropanol (Fisher) and 200 µl RNA precipitation solution (1•2 M sodium chloride and 0•8 M sodium citrate sesquihydrate) followed by centrifugation at 20 000 g. The resulting pellet was washed twice with 75 % ethanol, air-dried and resuspended in 50 µl nuclease-free water. The concentration and quality were verified spectrophotometrically (NanoDrop ND-1000 Spectrophotometer; Thermo Scientific) and by agarose gel electrophoresis to visualise the presence of 18S and 28S ribosomal subunits. Extracts were stored at -70°C until complementary DNA (cDNA) synthesis.
6
RNA Extraction Using TRIzol and miRNeasy
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Total RNA was extracted using both TRIzol (Invitrogen, Carlsbad, CA, United States) and the miRNeasy Mini Kit (QIAGEN, Valencia, CA, United States) according to the manufacturers’ protocols, with some modifications. Each sample was resuspended in 300 μl TRIzol reagent (Invitrogen). Resuspension was homogenized for 30 s using a Kontes Pellet Pestle Cordless Motor (Sigma-Aldrich, St. Louis, MO, United States) and placed on ice for 30 s, the steps being repeated 5 times for full homogenization. Then, an additional 700 μl TRIzol was added to the samples and incubated at room temperature for 5 min. 1-bromo-3-chloropropane (200 μl, Sigma-Aldrich) was then added to each sample, followed by incubation for 3 min. Next, the samples were centrifuged at 13,499 × g for 15 min at 4°C using a centrifuge (Smart R17 plus, Hanil Science, Incheon, South Korea). The supernatant (approximately 750 μl) of each sample was mixed with 750 μl of 100% isopropanol. The mixed samples were incubated at -20°C for 2 h. The columns of miRNeasy Mini Kit (QIAGEN) were additionally used to purify the small RNA-enriched RNA and clean RNA according to the manufacturer’s protocol.
7
RNA Extraction from Brain Tissue
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A 200-μL aliquot of the brain homogenate was combined with 800 μL of TRIzol (Thermo Fisher Scientific, Waltham, MA) and 200 μL 1-bromo-3-chloropropane (Sigma-Aldrich, St. Louis, MO), mixed, and centrifuged at 4 °C at 16,000×g for 15 min. The aqueous phase was added to QIAshredder columns (Qiagen, Valencia, CA) and centrifuged at 21,000×g for 2 min to fragment any remaining genomic DNA (gDNA) in the sample. The flow-through was combined with an equal volume of RLT buffer (Qiagen, Valencia, CA) with 1% β-mercaptoethanol, and RNA was extracted using AllPrep DNA/RNA 96 Kit as described by the manufacturer (Qiagen, Valencia, CA) including additional on-column DNase 1 treatment to remove gDNA. RNA purity and concentration was determined by spectrophotometry. RNA integrity was analyzed using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Equal amounts of RNA (400 ng) were used as a template for NGS library preparation.
8
Organoid RNA Extraction and RT-qPCR
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Organoids were harvested in a dark room, maintaining a controlled luminous intensity ranging from 1.6 to 5 lx, as measured using the BH1750 light sensor (Adafruit, NYC, USA). Furthermore, the entire lysis procedure was performed within a time frame not exceeding 5 minutes. At least 6 organoids per sample were washed with phosphate-buffered saline (PBS), and were homogenized using a 1 ml insulin syringe in 300 μl RNA Blue Reagent (an analogue of Trizol) (Top-Bio). After the addition of 1-bromo-3-chloropropane (Sigma-Aldrich) and centrifugation (21000 g, 15 minutes, 4°C), the cell lysates were separated into three layers. The top aqueous layer containing RNA was transferred to a new tube and precipitated by the addition of isopropanol, followed by 10 minutes incubation on ice and centrifugation (18000 g, 10 minutes, 4°C). RNA pellets were washed with 70% ethanol (8500 g, 10 minutes, 4°C), air-dried, solubilized in PCR grade water (Top-Bio). For reverse transcription quantitative real-time PCR (RT-qPCR), after the organoids were homogenized using a 1 ml insulin syringe in 300 μl RNA Blue Reagent, Direct-zolTM RNA Microprep kit (Zymo Reseach) was used according to the manufacturer’s instructions and as described previously.45 (link)
9
Measuring Inflammatory Markers in Liver of HFD/STZ Rats
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Elevated levels of inflammatory markers in liver, including interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), are believed to be associated with diabetes progression [34 (link), 35 (link)]. Therefore, the expression levels of IL-6 and TNF-α in liver in HFD/STZ rats were measured using RT-PCR. The total RNA in liver was isolated using TRIzol (Thermo Fisher, Carlsbad, CA, USA) followed by 1-bromo-3-chloropropane (Sigma-Aldrich, St. Louis, Mo, USA) extraction. The extracted messenger RNA (2 µg per sample) was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. RT-PCR was performed using TaqMan assay primer/probe (Roche) on a LightCycler 480 system. The signal intensity was normalized to β-actin. The primers used in this study are shown below.
10
RNA Extraction from Colonized Scaffolds
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Colonized bilayer scaffolds were snap-frozen at −80 °C. The samples were incubated with 1000 µL of pre-cooled Qiazol (Qiagen, Hilden, Germany) and disintegrated using a tissue lyser (Qiagen) at 50 Hz for 2 × 5 min. After incubating for 5 min at RT, 200 µL of 1-bromo-3 chloropropane (Sigma-Aldrich) was added. Centrifugation of the specimens for 15 min at 4 °C with 12,000× g followed, with the aim of obtaining a pure supernatant containing RNA which was pipetted on Qiashredder columns (Qiagen). Isolation of RNA followed the RNeasy Mini kit protocol (Qiagen) with an on-column DNase digestion. The Nanodrop ND-1000 spectrophotometer (Peqlab, Biotechnologie GmbH, Erlangen, Germany) was used to determine quantity and purity (260/280 absorbance).
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