HLA-DR and -DQ Typing by DNA-RFLP Analysis
M. Kam, Jeffrey L. Bidwell in Handbook of HLA TYPING TECHNIQUES, 2020
RFLP analysis was first applied to the investigation of HLA class II polymorphism by Wake et al.30 The technique (Figure 1) involves hybridization of labeled, single-stranded DNA sequences (probes) to homologous single-stranded DNA sequences (targets) within genomic DNA. Prior to hybridization, genomic DNA is treated by digestion with a restriction endonuclease; endonucleolytic fragments are then separated according to size by agarose gel electrophoresis and are transferred to a solid support by capillary or vacuum blotting. Although originally advocated as a technique with only a supplementary role in tissue typing, subsequent studies have shown that RFLP analysis permits the accurate genotyping of most DR, DQ, and Dw specificities.1-4-11-12-23-28 These investigations have shown that the correlation between RFLP and phenotypically determined DR, DQ, and Dw specificities varies according to the choice of restriction endonuclease employed.
Cellular and Molecular Mechanisms of Ischemic Acute Renal Failure and Repair
Robin S. Goldstein in Mechanisms of Injury in Renal Disease and Toxicity, 2020
The pathological features of necrosis are very different from those of programmed cell death (Searle, et al., 1982). The latter has been called “apoptosis”, a term coined by Kerr et al., who attributes the derivation of the term to Professor James Cormack of the University of Aberdeen (Kerr, et al., 1972). “Apoptosis” is a Greek term to describe “dropping off’ or “falling off’ of petals from a flower or leaves from a tree. Pathologically, the early stages of apoptosis are characterized by disappearance of microvilli, chromatin condensation at the periphery of the nucleus, condensation of cytosolic components, breakdown of epithelial desmosomal attachments, followed by cell surface protuberances, and fragmentation of the nucleus. Ultimately there is blebbing off of cell surface protuberances containing condensed cytosol and occasional nuclear fragments, to generate spherical or ovoid “apoptotic bodies” (Wyllie, et al., 1980) which can be phagocytosed by macrophages or epithelial cells. Apoptosis histologically is often associated biochemically with cleavage of double-stranded DNA at the linker regions between nucleosomes, resulting in fragments of approximately 180 to 200 bp (Kerr and Harmon, 1991). On agarose gel electrophoresis there is a characteristic ladder pattern. The endonuclease responsible for this biochemical correlate of apoptosis has not been well characterized. A candidate enzyme is an endonuclease that is activated by calcium in thymocytes (McConkey, et al., 1988). Increases in cellular calcium concentration can induce apoptosis (Kizaki, et al., 1989).
Glutathione and the Regulation of Apoptosis in the Nervous System
Christopher A. Shaw in Glutathione in the Nervous System, 2018
Although morphological criteria are its most consistent features, no one criterion defines cells as apoptotic. Therefore, to address whether glutathione depletion in embryonic cortical neurons leads to apoptosis or necrosis, several criteria were considered. Necrotic death is characterized by an early loss of membrane integrity and cell and organelle swelling. In vivo, membrane breakdown leads to a release of cytoplasmic contents and a fulminant immune response, including polymorphonuclear lymphocytes. DNA is degraded randomly, leading to a continuous smear upon agarose gel electrophoresis. Apoptosis, on the other hand, is characterized morphologically by chromatin condensation and nuclear and cytoplasmic shrinkage. In vivo, the cell compartmentalizes into many small, membrane-bound vesicles, which can be quietly digested by macrophages or neighboring cells. Loss of membrane integrity is a late event, and DNA is degraded into multiples of oligonucleosomal fragments of approximately 200 base pairs (with some exceptions). In some cases, apoptotic death requires RNA and DNA synthesis. This observation has fueled the notion that this form of death is “active” and requires the synthesis of specific “death proteins.”
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
Agarose gel electrophoresis was done using 7 × 10 cm Mini Sub cell Gel electrophoresis apparatus (Bio-Rad, Hercules, CA) as described in a manual (Green and Sambrook 2012). The 1% agarose gel was prepared by adding 1 g of agarose powder in 100 mL TAE (tris acetate EDTA) and boiled until the powder is dissolved. The agarose solution was cooled down to about 60 °C before pouring into gel tray that has been placed in an eight welled comb and bordered with a gel casting gates within the gel electrophoresis apparatus. The gel was allowed to solidify at room temperature and gently the comb and gates were removed. TAE buffer was poured into the electrophoresis apparatus to submerge the solidified gel. DNA ladder of 100 bp was used as DNA marker (Promega, Madison, WI). The DNA ladder marker was prepared by mixing 10 µL markers and 2 µL of 6× loading dye. Other wells were loaded with 10 µL of RT PCR products that had been premixed with 2 µL of loading dye. Gel electrophoresis was run at 80 V for 35 minutes. After electrophoresis, the gel was transferred from the electrophoresis apparatus and submerged into ethidium bromide staining solution for 15–30 minutes. The gel was destained with distilled water for 15 minutes and placed in UV transilluminator to visualize the nucleic acid bands.
Induced mutation in Agaricus bisporus by gamma ray to improve genetic variability, degradation enzyme activity, and yield
Published in International Journal of Radiation Biology, 2021
Tayebeh Harfi, Motallebi-Azar Alireza, Rasouli Farzad, Zaare-Nahandi Fariborz
Non-mutant genotype (control) and 15 superior variants were grown in 25ml of malt extract dextrose broth and incubated at 25° C for 15days for the growth of mycelia. Then mycelia were removed from the liquid medium, rinsed with sterile distilled water, and dried with filter paper. The ZR Fungal/Bacterial DNA KitTM procedure was used to extract total genomic DNA (Du et al. 2011). To determine the concentration of DNA in the original sample, DNA concentration absorbance was measured at 260nm using a Smart SpecTM Plus (Biorad, USA). DNA quality was examined by 0.8% agarose gel electrophoresis. Original DNA solutions were diluted to 25ng/µl with TE buffer, confirmed by visible spectrophotometry using ultraviolet (UV) absorption on a Smart SpecTM Plus (Bio-rad, USA) and stored at −20 °C until use.
Use of Decellularized SMILE (Small-Incision Lenticule Extraction) Lenticules for Engineering the Corneal Endothelial Layer: A Proof-of-Concept
Published in Current Eye Research, 2023
Swatilekha Hazra, Jacquelyn Akepogu, Supriya Krishna, SriRavali Pulipaka, Bhupesh Bagga, Charanya Ramachandran
Lenticules, at the end of the incubation period in the different media, were dried at 60 °C for 2 hrs and the dry weight of the lenticules was measured. Dried lenticules were chopped and suspended in 200 µl of lysis buffer containing 1 M Tris pH 8, 10% SDS, and 0.5M EDTA pH 8. After 10 mins of incubation at room temperature, 20 mg/ml Proteinase K (Genei, India) was added and incubated at 56 °C for 2 hrs. 10 µl of 5M NaCl and equal proportions of isopropanol were added and incubated at 4 °C for 10 mins and centrifuged at 12,000 rpm for 15 mins. The supernatant was removed followed by resuspension of the pellet in 70% ethanol and centrifugation at 12,000 rpm for 5 mins. Ethanol was discarded and the air-dried pellet was resuspended in TE buffer (pH 8). DNA was quantified using NanoDrop (Thermo Scientific, USA) and the values were used to calculate DNA concentration per milligram weight of lenticules (N = 3). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was performed on the data using GraphPad Prism V9.0. Significance was set at p < 0.05. Agarose gel electrophoresis was used to visualize the genomic DNA.
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