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Agarose gel

Anca Macovei, Mattia Donà, Daniela Carbonera, Alma Balestrazzi
DNA diffusion assay is a simple, sensitive and reliable technique which allows the assessment of programmed cell death (PCD) or necrosis events based on nuclear morphology. It consists in isolating nuclei from plant material, which are then embedded in agarose and subjected to lysis in alkaline buffers. Under these conditions, and due to the presence of abundant alkali-labile sites in the DNA, small pieces of DNA diffuse in the agarose gel giving a specific halo appearance when stained with fluorescent dyes like DAPI (4',6-diamidino-2-phenylindole)...
2018: Methods in Molecular Biology
Fernando Domínguez, Francisco Javier Cejudo
Programmed cell death (PCD) is a key process for the development and differentiation of multicellular organisms, which is characterized by well-defined morphological and biochemical features. These include chromatin condensation, DNA degradation and nuclear fragmentation, with nucleases and proteases playing a relevant function in these processes. In this chapter we describe methods routinely used for the analysis of hallmarks of developmentally regulated PCD in cereal seed tissues, which are based on agarose and polyacrylamide gel electrophoresis, in situ staining of DNA fragmentation, and cell-free assays of relevant enzymatic activities...
2018: Methods in Molecular Biology
Chun-Yung Huang, Chia-Hung Kuo, Chia-Hsin Lee
In this study, we extracted fucoidan from compressional-puffing-pretreated Sargassum crassifolium by hot water. The crude extract of fucoidan (SC) was degraded by various degradation reagents and four low-molecular-weight (LMW) fucoidans, namely SCO (degradation by hydrogen peroxide), SCA (degradation by ascorbic acid), SCOA (degradation by hydrogen peroxide + ascorbic acid), and SCH (degradation by hydrogen chloride) were obtained. The degradation reagents studied could effectively degrade fucoidan into LMW fucoidans, as revealed by intrinsic viscosity, agarose gel electrophoresis, and molecular weight analyses...
January 11, 2018: Marine Drugs
Mark A Toleman
S1 pulsed field gel electrophoresis (PFGE) is a method to separate the bacterial chromosome(s) from plasmid nucleic acids. When combined with ethidium bromide staining and UV visualization this method is excellent at assessing the number of plasmids in individual bacterial strains. It is also good at approximating the true size of each individual plasmid when run against a DNA molecular marker. However, downstream applications such as: the location of individual resistance genes on individual plasmids or the chromosome are hampered by very poor transfer of large DNA molecules from agarose gels to adsorbent nylon or nitrocellulose membranes...
2018: Methods in Molecular Biology
Shuai-Mei Liu, Meng-Xia Ni, Ming-Chao Zhang, Pei-Ran Zhu, Qiu-Yu Wu, Wei-Jun Jiang, Jing Zhang, Wei-Wei Li, Xin-Yi Xia
Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominant pigmentary genodermatosis, which is characterized by a mixture of hyperpigmented and hypopigmented macules on the dorsal of the hands and feet, and on the face presented like freckle. Identification of RNA-specific adenosine deaminase 1 (ADAR1) gene results in DSH. This study was mainly to explore the pathogenic mutation of ADAR1 gene and provide genetics counselling and prenatal diagnostic testing for childbearing individuals.Mutational analysis of ADAR1 gene was performed by polymerase chain reaction (PCR) and electrophoretic separation of PCR products by 1...
December 2017: Journal of Genetics
Serpil Demirağ, Erdoğan Malatyalı, Sema Ertuğ, Hatice Ertabaklar
OBJECTIVE: Trichomonas vaginalis is an anaerobic protozoon and the most common non-viral sexually transmitted pathogen. The present study was designed to determine the genotypes of T. vaginalis using polymerase chain reaction (PCR)-restriction fragment length polymorphism of actin gene. METHODS: A total of 20 isolates from symptomatic females isolated and cryopreserved at Adnan Menderes University, Research and Training Hospital Parasitology Laboratory were included...
December 2017: Türkiye Parazitolojii Dergisi
J Fu, F Lai, Y Chen, X Wan, G Wei, Y Li, H Xiao, X Cao
BACKGROUND: Carney complex (CNC) is an extremely rare, multiple endocrine neoplasia syndrome that occurs in an autosomal dominant manner. Mutations in PRKAR1A have been reported to be a common genetic cause of CNC. METHODS: In this study, we reported a Chinese pedigree of CNC that manifests mainly as spotty skin pigmentation and primary pigmented nodular adrenocortical disease. Whole blood samples of this pedigree were collected for DNA/RNA analysis. Polymerase chain reaction (PCR) and reverse-transcription polymerase chain reaction analyses were performed to amplify the 11 exons and adjacent introns of PRKAR1A...
January 9, 2018: Journal of Endocrinological Investigation
Samira Sedehi, Hadi Tabani, Saeed Nojavan
In this work, polypropylene hollow fiber was replaced by agarose gel in conventional electro membrane extraction (EME) to develop a novel approach. The proposed EME method was then employed to extract two amino acids (tyrosine and phenylalanine) as model polar analytes, followed by HPLC-UV. The method showed acceptable results under optimized conditions. This green methodology outperformed conventional EME, and required neither organic solvents nor carriers. The effective parameters such as the pH values of the acceptor and the donor solutions, the thickness and pH of the gel, the extraction voltage, the stirring rate, and the extraction time were optimized...
March 1, 2018: Talanta
Yao Liu, Lin Jia, Shu-Man Jiang, Dong-Yun Chen, Jiang-Shun Song, Jian Xu
BACKGROUND: Although globus pharyngeus is not rare in clinical practice, little is known about its associated gene polymorphism. We investigated the association between the SLC6A4 polymorphism and globus pharyngeus and its response to treatment with antidepressants. METHODS: A total of 84 patients were diagnosed with globus pharyngeus according to Rome III, and 160 healthy controls were genotyped for the SLC6A4 polymorphism using polymerase chain reaction amplification and agarose gel electrophoresis...
January 8, 2018: Digestion
Mahmoud Shekari Khaniani, Parisa Aob, Mohammadreza Ranjouri, Sima Mansoori Derakhsan
Background/aim: Huntington disease (HD) is a progressive adult-onset neurodegenerative disorder presenting an autosomal dominant inheritance. Since there is no information on the prevalence of HD in northwestern Iran, the aim of the present study was to determine the prevalence of HD and the number of CAG trinucleotide repeats in the population of northwestern Iran.Materials and methods: Genomic DNA was extracted from whole blood by the salting-out method. DNA samples were analyzed to determine the number of CAG trinucleotide repeats of the HD gene...
December 19, 2017: Turkish Journal of Medical Sciences
Leif Wide, Karin Eriksson
BACKGROUND: FSH, LH, and TSH are glycoprotein hormones secreted from the pituitary as fully and low-asparagine-glycosylated hormones. These glycoforms of the hormones exist as a large number of isoforms varying in their glycan contents of terminal anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU). Due to the immense heterogeneity and the low concentrations in serum it has been a challenge to develop reliable analytical methods to measure and characterize the circulating glycoforms of these hormones...
January 4, 2018: Upsala Journal of Medical Sciences
Jun-Wei Ye, Qin Li, Xiang-Yu Tian, Lei Bao, Hong-Fang Wang, Jian-Ping Ge
Premise of the study: To investigate a more detailed evolutionary history of Lindera obtusiloba (Lauraceae) and other Lindera species, polymorphic low-copy nuclear primers were developed. Methods and Results: Unigenes of the L. obtusiloba transcriptome greater than 800 bp in length were randomly chosen for initial design of 168 primers. Agarose gel electrophoresis and Sanger sequencing were used to select low-copy nuclear genes. Twenty-seven primers were obtained and were used to investigate genetic diversity in 90 individuals from 24 populations...
December 2017: Applications in Plant Sciences
Wenlong Carl Chen, Robyn Kerr, Andrew May, Babongile Ndlovu, Anelisa Sobalisa, Sanelisiwe T Duze, Lavania Joseph, Christopher G Mathew, Chantal Babb de Villiers
Long-term storage of whole blood can affect the integrity of DNA if it is not done under optimal conditions. The aim of this study was to determine whether long-term storage (2-19 years) of whole blood samples at -30°C had a negative effect on the quality or quantity of genomic DNA that could be recovered at extraction. Genomic DNA was isolated from 2758 whole blood samples collected in 4 mL EDTA vacutainers from 1997 to 2012. DNA was extracted using the Qiagen® FlexiGene® DNA kit. The average storage duration at -30°C was 12 years...
January 3, 2018: Biopreservation and Biobanking
Norbert W Lutz, Monique Bernard
We recently suggested a new paradigm for statistical analysis of thermal heterogeneity in (semi-)aqueous materials by 1H NMR spectroscopy, using water as a temperature probe. Here, we present a comprehensive in silico and in vitro validation that demonstrates the ability of this new technique to provide accurate quantitative parameters characterizing the statistical distribution of temperature values in a volume of (semi-)aqueous matter. First, line shape parameters of numerically simulated water 1H NMR spectra are systematically varied to study a range of mathematically well-defined temperature distributions...
December 21, 2017: Journal of Magnetic Resonance
Miguel A Lopez, Randi M Mackler, Matthew P Altman, Kristine E Yoder
The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer...
December 8, 2017: Journal of Visualized Experiments: JoVE
Meng-Wei Lin, Jai-Sing Yang, Chi-Cheng Lu, Chingju Lin, Sheng-Chu Kuo, Fuu-Jen Tsai, Miau-Rong Lee
The present study aimed to investigate the biological effects of the new compound 2‑phenyl‑4‑quinolone (YT‑1) on human leukemia cells. Cell viability was determined by propidium iodide (PI) exclusion method followed by flow cytometry. Our results showed that YT‑1 inhibited the cell viability and resulted in morphologic changes to the U937, HL‑60 and K562 cells, respectively. Among them, U937 cells were the most sensitive cell line. On the contrary, YT‑1 had no cytotoxic effects on human fetal skin fibroblast WS1 cells...
December 20, 2017: Oncology Reports
Nianhui Zhang, Dong Yu, Xiaofeng Zhu
The practice of RNA isolation in undergraduate experimental courses is rare because of the existence of robust, ubiquitous and stable ribonucleases. We reported here modifications to our original protocol for RNA isolation from plant tissues, including the recovery of nucleic acids by ethanol precipitation at 0 °C for 10 min and the assessment of RNA quality by visualizing the banding profile of the separated RNAs on a standard nondenaturing agarose gel to shorten the duration of the whole procedure and simplify the operation...
December 29, 2017: Biochemistry and Molecular Biology Education
K Moubri, J Kleuskens, J Van de Crommert, N Scholtes, T Van Kasteren, S Delbecq, B Carcy, E Précigout, A Gorenflot, Th Schetters
Soluble parasite antigens (SPA) in supernatants of in vitro cultures of Babesia canis can be used to vaccinate dogs against virulent B. canis infection. The moment that immunity becomes apparent coincides with the appearance of antibodies against SPA in the serum of the vaccinated animals. This so-called vaccination-challenge serum (VC-serum) was used to precipitate antigens from B. canis culture supernatants in agarose gels. This antigen preparation was then used to analyse the reactivity of sera from vaccinated dogs on western blots...
January 15, 2018: Veterinary Parasitology
Qimeng Li, Baicheng Yi, Zhihui Feng, Runsha Meng, Cheng Tian, Qiong Xu
OBJECTIVES: Ten-eleven translocation 1 (TET1) is a DNA methylcytosine (mC) dioxygenase discovered recently that can convert 5-mC into 5-hydroxymethylcytosine (5hmC). We previously reported that TET1 promotes odontoblastic differentiation of human dental pulp cells (hDPCs). The gene encoding the family with sequence similarity 20, member C (FAM20C) protein, is a potential TET1 target and showed demethylation during odontoblastic differentiation of hDPCs in our previous study. This study aimed to explore whether TET1-mediated hydroxymethylation could activate the FAM20C gene, thereby regulating hDPC differentiation...
December 25, 2017: Cell Proliferation
Séverine A Degrelle, Thierry Fournier
It is now demonstrated that the sex-specific maternal-placental-fetal interaction plays an important role in placental functions and pathologies. Determination of fetal-sex may therefore be an important consideration in studies using placenta samples. In this present study, we describe a simple, fast, and cheap protocol, which allows the fetal-sex determination of placental tissues from various starting materials (villi or formalin-fixed, paraffin-embedded (FFPE) tissues, isolated cytotrophoblasts or cellular debris from whole cell lysates, and cDNA) by a single duplex PCR reaction followed by agarose gel electrophoresis...
January 2018: Placenta
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