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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
Following isolation of DNA from the tissue or cells of interest, a fundamental process for DNA methylation analysis is the treatment of DNA with sodium bisulphite. This treatment converts unmethylated cytosine nucleotides to uracil. Following PCR amplification (discussed above), the uracil nucleotide is then converted into thymine. Cytosines that are methylated, however, are protected during this process, and therefore do not undergo any changes (see Figure 6.1, Chapter 6). This process therefore enables methylated and unmethylated cytosine residues to be easily distinguished during subsequent ‘downstream’ analysis, described below, and shown in Figure 2.4.
Pharmacology of Local Anesthetics
Published in Pamela E. Macintyre, Stephan A. Schug, Acute Pain Management, 2021
Pamela E. Macintyre, Stephan A. Schug
Because of its rapid onset, rapid metabolism, and short duration of action, chloroprocaine has been primarily used in obstetric epidural analgesia or regional anesthetic techniques for day surgery (Tonder et al, 2020). Neurotoxicity, with motor and sensory deficits, has followed accidental subarachnoid injection; the antioxidant sodium bisulfite in the anesthetic solution has been implicated as the cause. This has been replaced by ethylenediaminetetraacetic acid, usually abbreviated as EDTA, in recent formulations.
Timolol
Published in Anton C. de Groot, Monographs in Contact Allergy, 2021
Out of 112,430 patients patch tested by the IVDK between 1993 and 2004, 332 had been tested with their own topical anti-glaucoma eye drops containing different ß-blockers because of suspected allergic contact dermatitis. 189 subjects were tested with timolol eye drops and there were 21 (11%) positive reactions. The patients were not tested with the active substance, but reactions to the (possible) adjuvants benzalkonium chloride, sodium EDTA and sodium bisulfite were excluded (3).
Liquid biopsy from research to clinical practice: focus on non-small cell lung cancer
Published in Expert Review of Molecular Diagnostics, 2021
Umberto Malapelle, Pasquale Pisapia, Alfredo Addeo, Oscar Arrieta, Beatriz Bellosillo, Andres F. Cardona, Massimo Cristofanilli, Diego De Miguel-Perez, Valeria Denninghoff, Ignacio Durán, Eloísa Jantus-Lewintre, Pier Vitale Nuzzo, Ken O’Byrne, Patrick Pauwels, Edward M. Pickering, Luis E. Raez, Alessandro Russo, Maria José Serrano, David R. Gandara, Giancarlo Troncone, Christian Rolfo
Genome-wide methylation is detected mainly by measuring the content of 5mC in the genome [85,95]. Almost all of the cfDNA methylation analysis methods depend on bisulfite sequencing, which transforms non-methylated cytosines to uracils after sodium bisulfite treatment while not altering methylated cytosines [85]. Following DNA sequencing, DNA methylation sites can be detected. Sodium bisulfite treatment would cause a degree of DNA degradation which may lead to loss of some critical information [96]. For example, WGBS-based methods produce the most comprehensive and high-resolution DNA methylome maps, but typically require sequencing to 30× coverage which is still expensive for routine analysis. Additionally, optimized approaches named single-cell reduced-representation bisulfite sequencing (scRRBS) and Methylated CpG tandems amplification and sequencing (MCTA-seq) aim at capturing CpG-enriched cfDNA fragments, which would lead to loss of some critical DNA methylation sites, reducing the sensitivity of the methods.
DNA methylation changes in promoter region of CDKN2A gene in workers exposed in construction environment
Published in Biomarkers, 2020
Isana Rodrigues Silva, Luiza Flavia Veiga Francisco, Cassia Bernardo, Marco Antônio Oliveira, Fernando Barbosa, Henrique César Santejo Silveira
After treatment with sodium bisulphite conversion, each sample was submitted to polymerase chain reaction (PCR) with biotin-labeled primers. PCR was performed in a volume of 50 µL containing 2 µL converted DNA, 25 µL HotStarTaq Master Mix (Qiagen), and 1 µL of each primer (forward and reverse) at a concentration of 10 uM. The primers for CDKN2A, MLH1, APC, RASSF1A and repetitive sequences ALU and LINE-1 with the respective genomic locations of the target CpG sites in the genome were showed in Supplemental Table S1. The primers used in pyrosequencing assays to genes CDKN2A, MLH1, ALU and LINE-1 were kindly given by Dr. Zdenko Herceg from the International Agency for Research on Cancer (IARC) and synthesised as previously described by Silva et al. (2019) and Vaissière et al. (2009), and the genes APC, RASSF1A by Byun et al. (2012). The PCR products were purified using Sepharose Beads on a PyroMark Vacuum Prep Workstation (Qiagen) according to the manufacturer’s protocol and were transferred to a pyrosequencer (PyroMark Q96 ID System, Qiagen). Pyrosequencing was performed using the DNA from the samples and control template DNAs (methylated and non-methylated DNA) were obtained from (Qiagen). The methylation results were expressed as a percentage of methylated cytosines divided by the sum of methylated and unmethylated cytosines. Non-CpG cytosine residues were used to verify the efficiency of bisulphite conversion.
UV–Vis spectroscopic quantification of residual acetone during the development of nanoparticulate drug delivery systems
Published in Pharmaceutical Development and Technology, 2019
Sergio M. Espinoza, Rocio Guadalupe Casañas Pimentel, Eduardo San Martin Martinez
In general, samples for each experimental run consisted of an acetone aqueous solution prepared with NaHSO3, which was then introduced into a 10 mL calibrated flask along with NaOH and 2% m/v vanillin solution (prepared in 25% v/v ethanol in water). Afterward, leveling was performed with a 0.5% m/v NaHSO3 solution, which was prepared considering the percentage of Na2S2O5 in the granular sodium bisulfite. Sample and blank were transferred to a screw cap test tube, heated in a water bath, and then cooled before performing measurements. In the case of the first central composite design, heating time (10 and 30 min), heating temperature (40 and 80 °C) and cooling time (10 and 30 min) were contemplated as factors and were assessed by performing all experimental runs according to the experimental design (n = 3 for axial and factorial points, six central points). Sodium hydroxide solution concentration, vanillin solution volume, and acetone solution concentration were fixed at 1 M, 1 mL and ca. 5000 µg/mL, respectively. For experimental runs corresponding to the temperature axial point (92 °C, local water boiling point), a reflux apparatus was employed instead of the water bath.