Ten individual gonad samples and three gill samples for gametogenetic stage of diploid and triploid oysters were used for the quantitative expression analysis. The tissues were grounded in 500 µL of TriReagent (Sigma‐Aldrich, Saint Quentin Fallavier, France). One hundred microliters of bromo‐3‐chloropropane (Sigma‐Aldrich) was then added, and the samples were vortexed before centrifugation (12 000 g , 10 min, 4 °C). The aqueous phase was retrieved and the total RNA extraction was performed using a NucleoSpin RNA Clean‐up Kit (Macherey‐Nagel, Hoerdt, France), following the manufacturer’s instructions. RNA concentration, purity, and integrity were checked by spectrophotometry (NanoDrop ND‐1000, Thermo Scientific, Waltham, MA, USA). To prevent genomic DNA contamination, RNA samples were treated with DNAse I (1 U·µL−1 of total RNA, Sigma). Then, the total RNA was reverse‐transcribed to prepare for the cDNA. Two hundred and fifty nanograms of total RNA from each sample was reverse‐transcribed using 200 U of MMuLV‐RT (Moloney Murine Leukemia Virus Reverse transcriptase, Promega, Charbonnières les bains, France) in the presence of 12 U of RNase inhibitor (RNasin, Promega), 5 mm of RNAse free dNTPs, and 100 ng of random primers in the appropriate buffer (Promega) at 37 °C during 90 min. cDNA was stored at −20 °C until further usage.
Bromo 3 chloropropane
Manufactured by Merck Group
Sourced in United States, Germany
Bromo-3-chloropropane is a chemical compound used in various laboratory applications. It serves as a solvent and intermediate in organic synthesis processes. The core function of this product is to facilitate chemical reactions and extraction procedures in controlled laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Tri reagent, by Merck Group (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Dnase 1, by Merck Group (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Rnasin rnase inhibitor, by Promega (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Promega (1 mentions)
Random primer, by Promega (1 mentions)
Nucleospin rna clean up kit, by Macherey-Nagel (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Mouse ribopure blood rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Dna free kit, by Thermo Fisher Scientific (1 mentions)
Minispin centrifuge, by Eppendorf (1 mentions)
Trizol reagent, by Molecular Research Center (1 mentions)
Rneasy mini column, by Qiagen (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
5 protocols using bromo 3 chloropropane
1
Gonad and Gill RNA Extraction
2
RNA Isolation from Mouse Blood and Brain, Human Leukocytes
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For mice samples, total RNAs were purified from the blood using the Mouse RiboPure-Blood RNA isolation kit (Life Technologies, Ambion), according to manufacturer’s recommendations, and from brain samples using the mirVana miRNA isolation kit (Life Technologies, Ambion) after mechanical grinding of the tissues. For human samples, leukocytes trapped on LeukoLOCK filters were lysed with TRI reagent (Ambion) and mixed with Bromo-3-chloro-propane (Sigma-Aldrich, St. Louis, MO, USA). After centrifugation, total RNA from the aqueous phase was precipitated with ethanol and then purified on spin cartridge. After washings, total RNAs were eluted with 0.1 mM EDTA. Both human and mice total RNA samples were subsequently submitted to DNase treatment (DNA-freeTM kit, Life Technologies, Ambion, Austin, TX, USA). RNA concentration was determined using a nanodrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
3
RNA Extraction from Neutrophils
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RNA was extracted from BMNs using TRI reagent (Sigma) as per the manufacturer's instructions. Isolated neutrophils, untreated or treated with NA for either 4 or 24 h, were collected, centrifuged, and the supernatant aspirated and stored for quantification of cytokines and myeloperoxidase (MPO). Cell pellets were then lysed for 5 min at room temperature using TRI reagent. 1‐Bromo‐3‐chloropropane (Sigma) was then added to each sample and shaken vigorously before being allowed to stand for 5 min. The mixture was then centrifuged at 500 g at 4°C for 15 min, and the aqueous phase was collected. 2‐Propanol (Sigma) was then added to each sample and allowed to stand for 10 min before being centrifuged for 10 min at 500 g at 4°C. The supernatant was then removed, and the sample was washed with 75% (v/v) ethanol. The supernatant was again removed, the pellet was dried for 10–15 min in a laminar flow hood, and the pellet was resuspended in RNase‐free water.
4
RNA Isolation from Tissue Samples
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Total RNA was isolated using the RNeasy Mini kit (Qiagen, Inc.), following the manufacturer's protocol and as described previously (33 (link)). Briefly, the tissue was homogenized in TRIzol reagent (Molecular Research Center, Inc.), and the aqueous and organic phases were separated by the addition of one volume of bromo-3-chloropropane (Sigma-Aldrich; Merck KGaA), followed by centrifugation in a MiniSpin® eppendorf centrifuge at 13, 800 × g for 15 min at 4°C. Next, 350 µl of 70% ethanol (Sigma-Aldrich; Merck KGaA) were added to all samples and each sample was applied to an RNeasy mini-column. The columns were washed by centrifugation at 735 × g for 2 min at room temperature with buffers containing guanidine and ethanol. In order to elute the RNA, RNase-free water (30 µl) was added directly onto the silica-gel membrane of the columns and each column was centrifuged for 1 min at 13, 800 × g at room temperature. The RNA was quantified in a SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, Inc.) by measuring absorbance at 260 nm and was stored at −85°C until use. The quality of each RNA sample was assessed on 2% formaldehyde denaturing agarose gels.
5
Total RNA Isolation Protocol
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Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. In brief, the tissue was homogenized in TRI reagent (Molecular Research Center, Cincinnati, OH, USA), and the aqueous and organic phases were separated by addition of one volume of bromo-3-chloropropane (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), followed by centrifugation at 13, 800 × g for 15 min at 4°C. Thereafter, 70% ethanol (350 µl) was added to each sample, which was then applied to an RNeasy minicolumn (Qiagen), followed by washing by centrifugation at 735 × g for 2 min at room temperature with buffers containing guanidine and ethanol. To elute the RNA, 30 µl RNase-free water was added directly onto the silica-gel membrane of each of the columns, which were then centrifuged for 1 min at 13, 800 × g at room temperature. The RNA was quantified by measuring the absorbance at 260 nm and stored at −85°C until use. The quality of each RNA sample was assessed on a 2% formaldehyde denaturing agarose gel.
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