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Pharmacology and Therapeutics of Dandelion in Gastrointestinal Disorders
Published in Megh R. Goyal, Preeti Birwal, Durgesh Nandini Chauhan, Herbs, Spices, and Medicinal Plants for Human Gastrointestinal Disorders, 2023
Zhu et al.29 evaluated the aqueous extract of dandelion on two gastric cell lines (SGC7901 and BGC823) along with one gastric epithelium cell line (GES-1) as the control. CV and colony formation assay were evaluated after the ribonucleic acid isolation using trizol reagent. The outcomes revealed that: CV was dose-dependent and was suppressed in both cell lines.The concentration (3 mg/mL) of the extract showed greater antiproliferative effect and inhibition of the cancerous cell invasion to the secondary phase.Total anticancer activity of the extract was mediated through long non-coding RNA-colon cancer-associated transcript1.
Human Gut Microbiota–Transplanted Gn Pig Models for HRV Infection
Published in Lijuan Yuan, Vaccine Efficacy Evaluation, 2022
RT-PCR for rotavirus gene segment 6 was used to screen samples for rotavirus. RNA was extracted from samples using TRIZOL LS (ThermoFisher Scientific) per the manufacturer's instructions. The mixture for cDNA synthesis included 2 µmol of primer 1581 and 11 µl of RNA. RNA was denatured at 95° C for 5 min then cooled to 4° C. The mixture for reverse transcription contained 4 µl of 5X buffer, 1µl 0.1 M DTT, 1 µl Superscript III reverse transcriptase (ThermoFisher Scientific), and 1 µl of RNase free water. The cDNA sample was combined with the mixture for reverse transcription and incubated at 50° C for 60 min then 70° C for 15 min and held at 4° C. One µl of RNaseH (New England Biolabs, Ipswich, MA) was added and the sample was incubated at 37° C for 20 min. The PCR mix contained 10 µl of buffer, 1 µl of MyTaq DNA polymerase (Bioline, Taunton, MA), 27 µl of ddH2O, 1µl of each 20 μM primer (158 [forward] and 15 [reverse]), and 10 µl of DNA. The sequence for primer 158 was GGC TTT AAA ACG AAG TCT TCG AC and that for primer 15 was GGT CAC ATC CTC TCA CTA. The sample was incubated at 95° C for 3 min followed by 35 cycles of 95° C 20 sec, 47.5° C 30 sec, and 72° C 90 sec. The final extension was at 72° C for 7 minutes. AttHRV was used as a positive control.
Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
This section consists of techniques for extracting RNA from different culture conditions or tissues (e.g., 2D vs. 3D) and the manipulation and detection of changes in gene expression. It is important to note that for extraction and handling of RNA, it may be advisable to have a small section of the lab bench cordoned off with reagents and pipettes only used for RNA handling. RNA is extremely sensitive to degradation, and RNAses are ubiquitous and very stable. Care should also be taken to prevent DNA contamination, especially from polymerase chain reaction (PCR) products, which may give false signal during quantitative reverse transcription (qRT) PCR measures of gene expression. The following protocols are separated by RNA extraction from 2D culture via lysis buffer and RNA extraction from 3D culture following liquid nitrogen pulverization. The procedure for the column cleanup is identical for both. Depending on the size of columns used, the amount of starting material should be adjusted to ensure the column is not overloaded. It should be noted that extraction of good-quality RNA is also possible without the use of columns by using TRI-reagent-based chemistry (such as TRIzol). However, on-column DNA removal may still be necessary for some applications.
The involvement of Nrf2/HO-1/cytoglobin and Ang-II/NF-κB signals in the cardioprotective mechanism of lansoprazole against cisplatin-induced heart injury
Published in Toxicology Mechanisms and Methods, 2023
Emad H. M. Hassanein, Fares E. M. Ali, Zuhair M. Mohammedsaleh, Ahmed M. Atwa, Mohamed Elfiky
Lansoprazole (LPZ) and CIS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lactate dehydrogenase (LDH; 1001260), alkaline phosphatase (ALP; MI41233), aspartate transaminase (AST; MI41264), creatinine kinase-MB (CK-MB; 1001055) kits were obtained from SPINREACT (Spain). Rats’ troponin-I (E-EL-R1253), Ang-II (E-EL-R1430), Ang 1–7 (E-EL-R1138), TNF-α (E-EL-R2856), and IL-6 (E-EL-R0015) ELISA kits were purchased from Elabscience (China). TRIzol was supplied by Thermo Fisher Scientific (USA). Total RNA extraction kits, cDNA synthesis kits, and PCR primers were purchased from Vivantis Technologies (Malaysia). Primary antibodies for NF-κB (sc-71675, dilution 1:1000), IκB (sc-1643, dilution 1:500), STAT-3 (sc-293151, dilution 1:1000), p-STAT-3 (sc-81523, dilution 1:1000), PPAR-γ (sc-271392, dilution 1:500), and β-actin (sc-47778, dilution 1:2000) were obtained from Santa Cruz Biotechnology (USA). Nrf2 (YPA1621, dilution 1:500), HO-1 (YPA1919, dilution 1:1000), and cytoglobin (YPA1394, dilution 1:500) primary antibodies were purchased from Biospes Company (China). All other chemicals and reagents were obtained from a certified local supplier.
Up-regulation of long non-coding RNA LOXL1-AS1 functions as an oncogene in cervical squamous cell carcinoma by sponging miR-21
Published in Archives of Physiology and Biochemistry, 2023
Trizol (Life Technologies) was used to extract total RNAs from the tissue samples from patients and in vitro cultivated cells. To remove genomic DNA, all RNA samples were digested with LookOut® DNA Erase (Sigma-Aldrich). Reverse transcriptions were performed using PrimeScript™ RT reagent kit (Takara) with total RNA samples as template, and qPCR assays were performed using SYBR® Premix Ex Taq™ II (Takara) with GAPDH as endogenous control to measure the expression levels of LOXL1-AS1 and RHOB mRNA. To quantify the level of mature miR-21 expression, all steps, including polyadenylation, RTs and qPCR assays were completed by All-in-OneTM miRNA qRT-PCR Detection Kit (Genecopoeia). All PCR reactions were repeated 3 times and 2−ΔΔCq method was used to calculate the fold changes of gene expression across samples.
The induction of aryl hydrocarbon receptor (AHR) in immune organs of developing chicks by polycyclic aromatic hydrocarbons
Published in Toxicology Mechanisms and Methods, 2018
A. R. Nisha, H. Hazilawati, M. L. Mohd Azmi, M. M. Noordin
Total RNA was isolated from bursa of Fabricius, spleen, and thymus tissues using Trizol reagent as per qiagen catalog number 79654. About 30–40 mg of tissue was added to 1 mL of Trizol in a cell culture tube that was precut at the 8 mL mark. Tissue samples were well homogenized with autoclaved probe. The tube was kept on ice throughout process. The homogenized sample was transferred to a shredder column and centrifuged at 15 000×g at room temperature for two minutes. The filter column was removed after centrifugation and a new cap was placed on the tube and centrifuged at 15 000×g for another three minutes. The supernatant was transferred to a 1.5 mL tube after centrifugation and incubated for five minutes at room temperature. Two hundred microliter of chloroform was added and vortexed well. The tube was incubated for 2–3 minutes at room temperature and centrifuged at 12 000×g for 15 min at 4 °C. The aqueous phase was transferred to 1.5 mL tube after centrifugation. Isopropanol 0.5 mL was added, and the mixture vortexed and incubated for 10 minutes at room temperature, later centrifuged at 12 000×g for 10 minutes at 4 °C. The isopropanol was removed after centrifugation using a suction apparatus, and then the pellet was washed with 1 mL of 80% ethanol. The contents in the tube were mixed well and then centrifuged at 10 000×g for seven minutes at 4 °C. The ethanol was removed by the vacuum apparatus after centrifugation. The pellet was air dried in the hood, and then dissolved in 50 µL of RNase free water. Then another 50 µL of RNase free water was added more to make a total of 100 µL.