China
Africa
Explore chapters and articles related to this topic
Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
Importantly, as mentioned above, RNA is much less stable than DNA and therefore presents some key methodological challenges. Most importantly, RNA degrading enzymes known as ribonuclease (RNase) can break down the RNA molecule into smaller fragments that are unusable for experiments. It is therefore recommended to always ensure good laboratory practice (i.e. always wearing clean gloves, a lab coat and cleaning all surfaces and equipment with RNase-inhibiting solution) and always use RNase-free plasticware and diethylpyrocarbonate (DEPC)-treated water when making relevant buffers and reagents.
The Modification of Histidine Residues
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
McCormick and co-workers42 have examined the reaction of diethylpyrocarbonate with pyridoxamine (pyridoxine)-5’-phosphate oxidase. The modification reaction was performed at pH 7.0 (0.1 M potassium phosphate containing 5% (v/v) EtOH) at 25°C generally in the presence of flavin mononucleotide (FMN). Figure 12 shows the time course for the modification of the oxidase under these experimental conditions. Also shown in Figure 12 is the dependence of the observed first-order rate constant on diethylpyrocarbonate concentration (panel B). The inset plot shows the reaction to be second-order with a rate constant of 12.5 M−1 s−1.
The Toxicology and Biological Properties of Organotin Compounds*
Published in Nate F. Cardarelli, Tin as a Vital Nutrient:, 2019
Triethyltin increased the reactivity of the sulfhydryl groups to 4,4′-dipyridyl disulfide instead of decreasing, as would be expected if these groups were involved in the binding site for triethyltin.133 Treatment with diethylpyrocarbonate also showed that reaction with triethyltin did not depend on the availability of histidine groups.133
Exogenous glutamine ameliorates diabetic nephropathy in a rat model of type 2 diabetes mellitus through its antioxidant and anti-inflammatory activities
Published in Archives of Physiology and Biochemistry, 2023
Maryam Nasri, Glavizh Adibhesami, Sina Mahdavifard, Esmaeel Babaeenezhad, Hassan Ahmadvand
At the first step, about 100–200 mg of the frozen left kidney samples were placed inside a microtube containing Trizol reagent and thereafter homogenised using an ultrasonic homogeniser (HIELSHER UP 200H, Germany). In the second step, homogenised samples were incubated for 5 min at 25°C. After adding 200 μl of chloroform to the tissue homogeneous, it was centrifuged for 15 min (12,000 × g, 4 °C). The two-phase solution was formed following centrifugation, in which 250 μl of its upper phase was transferred to another sterile microtube. At this stage, 250 μl of isopropyl alcohol was added to the microtube and centrifuged for 15 min (12,000 × g, 4 °C). After that, RNA sediment was seen in centrifuged microtubes. After sedimentation, RNA sediment was washed with 75% ethanol and centrifuged (7500 rpm). At the last stage, the precipitated RNA was dried under sterile conditions. and dissolved into 30–40 μl of the diethyl pyrocarbonate–H2O (DEPC–H2O).
FASN Targeted by miR-497-5p Regulates Cell Behaviors in Cervical Cancer
Published in Nutrition and Cancer, 2022
Haiyan Zhang, Runmei Wang, Xuerui Tang, Jun Li, Jie Li, Mingxin Wang
Total RNA was extracted by TRIzol method (Takara Biomedical Technology [Dalian] Co., Ltd). Using reverse transcription kit (DRR047S, Takara Biomedical Technology [Dalian] Co., Ltd), the RNA was reversed into complementary DNA (cDNA). Next, the obtained cDNA was diluted with diethyl pyrocarbonate (DEPC). The qRT-PCR reaction system consisted of the following components: 5 μl of SsoFast EvaGreen Supermix (1708882, Bio-Rad, CA, USA), 0.5 μl of forward primer (10 μM), 0.5 μl of reverse primer (10 μM) and 4 μl of cDNA. The primers were synthesized by Shenzhen BGI Co., Ltd. (Shenzhen, Guangdong, China) (Table 1). U6 or GAPDH was used as internal reference. 2−ΔΔCt value was performed for the relative expression of target genes. The assay was repeated three times independently.
microRNA-17-5p downregulation inhibits autophagy and myocardial remodelling after myocardial infarction by targeting STAT3
Published in Autoimmunity, 2022
Bo Chen, Yingjun Yang, Jinbo Wu, Jianjiang Song, Jia Lu
Total RNA was extracted from myocardial tissues by Trizol one-step method according to the instructions of TRIzol (Invitrogen Inc., Carlsbad, CA) reagent. RNA was dissolved by ultrapure water treated with diethyl pyrocarbonate (DEPC). The absorbance at the wavelength of 260 nm and 280 nm was detected by an ultraviolet-visible spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, USA). The quality of total RNA was checked and the concentration of total RNA was adjusted. The experimental conditions were as follows: at 70 °C for 10 min, ice-bath for 2 min, at 42 °C for 60 min, and at 70 °C for 10 min. The complementary DNA obtained from reverse transcription was preserved at −80 °C temporarily. RT-qPCR was operated by the TaqMan probe method in accordance with the instructions of the kits (Fermentas Inc., Vilnius, Lithuania). The RT-qPCR was performed on ABI 7500 instruments (ABI, Foster City, CA, USA) with U6 as an internal control. The relative expression was determined with the 2−ΔΔCT method. Primer sequences are illustrated in Table 1.