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    • Introduction Nucleic acids are polyanionic and their negativ

    2020-08-18

    Introduction Nucleic acids are polyanionic and their negatively charged nucleotides allow the binding of cationic dyes, such as acridine orange (3,6-dimethylaminoacridine). Acridine orange (AO) was first synthesized in 1889, but its ability to bind nucleic acids was only reported in 1940, with greater improvement by 1950´s (von Bertalanffy and Bickis, 1956). Since then, a number of biological applications have been assigned to this fluorophore (Robbins and Marcus, 1963). AO interacts with double-stranded DNA by intercalation, while the single-stranded RNA interacts by ionic interactions and dye stacking, resulting in different fluorescence with maximum emission at λ = 530 nm and 640 nm for DNA and RNA, respectively (McMaster and Carmichael, 1977). The basophilic AO binds to other anions, such as carboxyl groups, from biological molecules other than nucleic acids. In fact, although it has long been used for staining DNA and RNA, the use of AO in tissue sections is hampered by the non-specific staining of other anionic groups, in particular those in the extracellular matrix.
    Materials and methods
    Results
    Discussion In this study, we used the ventral prostate gland as a model system to demonstrate the applicability of a combination of AO and FG staining for the simultaneous staining of DNA and RNA, and variations of RNA content in different physiological conditions. The direct staining of VP sections with AO resulted in diffuse staining of the tissue, with marked labeling of secretory products and the extracellular matrix. These are interfering factors in applying the classical procedure to tissue sections, as compared to isolated Tenatoprazole or cell nuclei (Darzynkiewicz, 1990). The proposed FG counterstaining was based on its specificity for total protein imino by guanidinegroups, and its use resulted in quenching of the AO fluorescence, both in the secretory products in the glandular lumen and in the extracellular matrix. Furthermore, we observed that the different ductal regions exhibited different levels of red fluorescence, which is compatible with the increased secretory activity in the intermediate and distal ductal regions, compared to the proximal region proposed by others (Lee et al., 1990; Nemeth and Lee, 1996). Still exploring the VP, we also demonstrated that castration promotes a reduction of red fluorescence (RNA), which is compatible with the reduced transcriptional and secretory activity. Knowing that the prostate function is also directly modulated by somatotrophic hormones such as insulin (Damas-Souza et al., 2010; Webber, 1981), we examined the RNA content as indicated by the red fluorescence in the VP of diabetic animals. We found a decreased RNA content in the epithelial cells, which was reversed by insulin administration, thus confirming the role of insulin in contributing to the maintenance of the functional state of the gland. We were also able to manipulate the metabolic status of the PC3 prostate cancer cells by exposure to different concentrations of glucose. As expected, there was a linear (R2 = 0.886) correlation between the glucose concentration in the culture medium and the amount of RNA extracted from the PC3 cells. Similarly, cultured cells showed increased red fluorescence after AO-FG staining, demonstrating a correlation between the metabolic state determined by biochemical measurement and the intensity of the AO red fluorescence.
    Introduction The separation and visualization of DNA by electrophoresis is associated with a variety of analytical and diagnostic assays. Visualization is carried out through different methods such as silver staining and various fluorescent dyes like ethidium bromide (EB) and SYBR Green I (SGI) [1], [2]. Although silver staining is very sensitive, it lacks selectivity and specificity, does not allow sample recovery and usually takes several hours. On the other hand, the use of fluorescent dyes allows analyte recovery and is more selective than silver staining, but requires instruments such as a transilluminator (UV or blue light) or fluorimeter for visualization [3]. Further, dyes such as EB require UV irradiation, which causes damage to both the DNA sample and the experimentalist [4], and uses visualization equipment. Finally, DNA labeling with radioactive isotopes is very sensitive, but is both hazardous and expensive, limiting its application in most laboratories [5], [6].