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Diethyl pyrocarbonate

Manufactured by Merck Group
Sourced in United States, Germany, Belgium

Diethyl pyrocarbonate is a chemical compound used in laboratory settings. It functions as a reagent to inactivate RNase enzymes, which are known to degrade RNA samples. This property makes diethyl pyrocarbonate a useful tool in RNA-related experiments and research.

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54 protocols using diethyl pyrocarbonate

1

RNase-free Experimental Procedures

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During all processes, precautions were taken to minimize the risk of RNase contamination. All working areas were treated with RNase inhibitors (RNase AWAY® Reagent, Ambion®). Glassware was heated at 180 °C overnight. Certified RNase free disposables were used during each step. Diethyl pyrocarbonate (DEPC) water was prepared by mixing 0.1% Diethyl pyrocarbonate (Sigma Aldrich, Diegem, Belgium) with ultrapure water, followed by incubating overnight at room temperature and autoclaving. All experiments were performed in compliance with the MIQE guidelines17 (link). All experiments were approved by the Ethical Committee of the Faculty of Veterinary Medicine and Bioscience-Engineering, Ghent University (no. EC2013/19) and carried out in accordance with the approved guidelines and legislation in force.
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2

Optimized Cryopreservation of Mouse Brain Sections for LCM

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For all experiments, reagents were of molecular biology grade. Buffers were pretreated overnight with diethylpyrocarbonate (Merck; 1 ml/l) and autoclaved or prepared using diethylpyrocarbonate-treated and autoclaved water as diluent. The working area was cleaned with RNaseZAP (Merck KGaA). To minimize possible adverse effects of fixation on RNA integrity and recovery, 0.5% formaldehyde in 0.1 M PBS (80 ml) was chosen for transcardiac perfusion of ChAT-ZsGreen mice, followed by ice-cold 20% sucrose in PBS (50 ml). The brains were snap-frozen on pulverized dry ice and stored at −80 °C until sectioned with a cryostat. Twelve-micrometer-thick sections containing the MS and the dorsal CPU (Atlas plates 20–26 of Paxinos; Bregma +1.34 to +0.62 mm) (38 ) were thaw-mounted onto PEN membrane glass slides (Membrane Slide 1 PEN, Carl Zeiss), air-dried in the cryostat chamber, and preprocessed for LCM as reported elsewhere (18 , 22 , 23 ). In brief, the slides were immersed sequentially in 50% EtOH (20 s), n-butanol:EtOH (25:1; 90 s) and xylene substitute:n-butanol (25:1; 60 s). Then, they were stored at −80 °C in slide mailers with silica gel desiccants or processed immediately for LCM.
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3

Reagents and Oligonucleotides for Telomere Assay

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Potassium
hexacyanoferrate(III) (99.98%),
potassium hexacyanoferrate(II) trihydrate (≥99.95%), ethylenediaminetetraacetic
acid (EDTA) ACS reagent (99.4–100.6%), 2-propanol anhydrous
(99.5%), sodium chloride (≥99.95%), acetic acid (≥99%),
Tween 20, Trizma base (99.9%) Trizma hydrochloride (≥99%),
methanol (≥99%), phenylmethanesulfonyl fluoride (PMSF), diethyl
pyrocarbonate (DCEP) (≥97%), dl-dithiothreitol (DTT)
(≥98%), glycine (≥99%), sodium dodecyl sulfate (SDS;
≥98.5%), and ddGTPs were purchased from Sigma-Aldrich (St.
Louis, MO). Acrylamide (40%), ammonium persulfate, tetramethylethylenediamine,
4× Laemmli sample buffer, and precision plus protein standard
were obtained from BioRad (Hercules, CA). Tris(2-carboxyethyl) phosphine
hydrochloride (TCEP), sulfuric acid optima (93–98%), Coomassie
Brilliant Blue G-250, HEPES buffer, detergent-compatible Bradford
assay kit, and RPMI-164 medium + 2.05 mM glutamine (Hyclone) were
purchased from Fisher Scientific (Fair-lawn, NJ). All synthetic oligonucleotides
were purchased from Integrated DNA Technology (IDT) (San Diego, CA)
with the following sequences:
TS30 (5′-S-S-(CH2)6-TTTTTTTTTTAATCCGTCGAGCAGAGTT-3′),
TS60 (5′-S-S-(CH2)6-TTTTTTTTTTAATCCGTCGAGCAGAGTTAGGGTTAGGGTTAGGGTTAGGGTTAGG-3′),
and
TSC (5′-S-S-(CH2)6-AAAAAAAAAATTAGGCAGCTCGTCTCAA-3′).
dNTPs were purchased
from Promega (Madison, WI, USA).
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4

Antimalarial Compounds Evaluation

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Mefloquine, chloroquine, artesunate, and diethylpyrocarbonate (DEPC) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Piperine (98%purity) was purchased from Wako Pure Chemical Co., Ltd. (Tokyo, Japan). Roswell Park Memorial Institute (RPMI) 1640, HEPES, and gentamicin were supplied by Gibco BRL Life Technologies (Grand Island, NY, USA). SYBR Green I was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Ethanol was purchased from Labscan Co. Ltd. (Bangkok, Thailand). All reference compounds and piperine were prepared as stock solutions of 10 mM and 3.5 mM in 50% ethanol, respectively.
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5

Porous Hydrogel Scaffold Fabrication

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Gelatin type A from porcine
skin (bloom 300), GA (25% solution in water), glycine, 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimidehydrochloride
(EDC), diethylpyrocarbonate, glycyrrhetinic acid, and collagenase
from Clostridium histolyticum in addition
to the chemicals that were used in the cytotoxicity studies: dimethyl
sulfoxide (DMSO), MTT, and trypan blue dye were purchased from Sigma,
St. Louis, Mo., USA. Acetone was purchased from Labscan, Dublin, Ireland.
Allicin was purchased from Jiangsu chiataiqingjiang Pharmaceutical
Co., Ltd., China. Spectra/Por dialysis membrane, 12 000–14 000
molecular weight cutoff, was purchased from Spectrum Laboratories
Inc., Rancho Dominguez, Canada.
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6

Molecular Mechanism of Apoptosis Induction

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4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid and trypsin were purchased from AMRESCO. Bovine serum albumin (BSA) was purchased from Sijiqing Hangzhou Bioengineering Company. Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from GIBCO BD. MTT, diethylpyrocarbonate, and ethidium bromide were purchased from Sigma. Dimethyl sulfoxide was purchased from Shanghai Ling Feng Chemical Co., Ltd. RNAiso Reagent, AMV reverse transcriptase, deoxyribonucleoside triphosphate (dNTP), Oligo(dT)18, Taq DNA polymerase, 100 bp DNA Marker, RNasin, and RNase free DNase I were purchased from Takara. From Shanghai Shenneng Gaming Biotechnology Co. Ltd. (Whitehouse Station, NJ, USA), 20×TBE, agarose, caspase-3 primer, caspase-8 primer, caspase-9 primer, and β-actin primer were purchased. C225 solution for infusion was purchased from Merck & Co, Germany. All other chemicals were commercially available and of analytical grade.
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7

Quantification of Katanin p60 Expression

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Cells were collected after transfected with plasmids or shRNAs for 48 h. At room temperature, total RNA was extracted with TRIzol (Thermo Fisher Scientific, Inc.) for 5 min, reacted with chloroform for 2–3 min, isopropanol for 10 min and 75% ethanol for 1 min sequentially, then RNA was dissolved in 0.1% diethyl pyrocarbonate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The concentration and purity of RNA were measured using a spectrophotometer (GE Healthcare, Chicago, IL, USA). Reverse transcription was performed using PrimeScript 1st strand cDNA Synthesis Kit (cat no. 6610A; Takara Biomedical Technology, Beijing, China), for 10 min at 25°C, 50 min at 42°C and 5 min at 85°C. qPCR analysis was performed using SYBR® Premix DimerEraser (Perfect Real Time; cat no. RR091A; Takara Biomedical Technology). The primer sequences were as follows: Katanin p60, forward, 5′-TAAACTGGACAGCACTCCCTTG-3′ and reverse, 5′-CCTGGTGAGGGTCTTCGTTC-3′; actin, forward, 5′TGACGTGGACATCCGCAAAG-3′ and reverse, 5′-CTGGAAGGTGGACAGCGAGG-3′. The thermocycling parameters were as follows: 94°C for 4 min; 35 cycles of 94°C for 20 sec, 60°C for 30 sec and 72°C for 30 sec. The relative expression of katanin p60 was obtained according to the 2−ΔΔCq method (19 (link)).
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8

Preparation and Immunostaining of Tissue Scaffolds

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The carriers were fixed in 4% formalin for 1 h, cut in half, and washed with phosphate-buffered saline. Scaffolds were automatized, dehydrated, and processed in standard paraffin wax (Vogel) using a Tissue-Tek VIP® Vacuum Infiltration Processor (Sakura) and the TES Valida Dispenser Unit (MEDITE), as described in Table 1. Serial sections (8–10 μm) were taken from the scaffolds at 100 μm intervals. Slides were deparaffinized, and different antigen retrievals were tested to find the ideal method for antigen-demasking before the immunostaining.
Low-temperature enzymatic antigen demasking was performed using pepsin, hyaluronidase, and proteinase K. Slides were either incubated with pepsin (0.1% in diethylpyrocarbonate; Sigma-Aldrich) for 30 min, hyaluronidase (0.02% in phosphate-buffered saline and 0.01% bovine serum albumin; Sigma-Aldrich) for 20 min, or proteinase K (5% in TE-Buffer pH 8; Roche) for 15 min at 37°C. Alternatively, heat-induced antigen retrieval was performed in a KOS microwave (Milestone) with Target Retrieval Solution pH 9 (Dako) at 93°C for 15 min. After blocking in H2O2 Block (ThermoScientific) for 10 min and UV Block (ThermoScientific) for 5 min, slides were exposed to anti-vimentin (0.078 μg/ml; Dako) for 30 min to detect hAMSCs on the carriers. Slides were developed using the UltraVision LP Detection System (ThermoScientific).
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9

Sampling and Extraction of Placental Tissues

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Women with euploidy pregnancies who attended KK Women’s and Children’s Hospital, Singapore, were recruited.
Chorionic villus samples from subjects at the first or early second trimesters of pregnancy were collected by chronic villus sampling (CVS). Placenta villi samples (fetal side) were collected from third trimester of pregnancy after delivery. All tissue samples were washed with diethylpyrocarbonate (Sigma-Aldrich, USA) treated water. For DNA analysis, tissues were stored at -80°C. For RNA analysis, tissues were incubated with RNAlater (Life Technologies, USA) at 4°C overnight, and then stored at -80°C. Genomic DNA extraction from tissues was performed with QIAamp DNA Mini Kit (QIAGEN GmbH, Germany), according to manufacturer’s instructions. Total RNA was extracted from frozen tissues using TRIZOL protocol (Life Technologies).
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10

Lentiviral Vector Production in HEK293FT Cells

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PAa lung adenocarcinoma cells were obtained from Peking University Health Science Center (Beijing, China), and the 293FT human embryonic kidney cell line was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Lentiviral vectors [pLLU2G-green fluorescent protein (GFP)], packaging systems (3rd generation lentivirus packing system) and negative control virus particles (pLP1, pLP2, pLP/VSV-G and pLLU2G) were obtained from Invitrogen (Thermo Fisher Scientific, Inc.) Lipofectamine® 2000 transfection reagent and One Shot® Stbl3™ chemically competent E. coli were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The QIAquick Gel Extraction Kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and diethylpyrocarbonate were all purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany).
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