RESUMEN
Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness.
Asunto(s)
Citometría de Flujo/métodos , Transfección/métodos , Sistemas CRISPR-Cas , ADN/genética , Electroporación , Terapia Genética/métodos , Proteínas Fluorescentes Verdes/química , Células HEK293 , Humanos , Células Jurkat , Plásmidos/química , ARN/genética , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Although 75% of suicides occur in low- and middle-income countries, few studies have examined suicidal behaviors among young people in these countries. AIMS: This study aimed to examine what individual characteristics were associated with suicidal plans and attempts among Mongolian youth and whether suicidal risks and behaviors varied by urban and rural locations. METHOD: Logistic regression analyses were utilized to investigate suicidal plans and attempts among 5,393 adolescents using the Global Student Health Survey - 2013. RESULTS: Adolescents who lived in urban areas were at higher risk for suicidal plans and behaviors than those who lived in rural areas; however, the patterns of suicidal risks were similar. Specifically, individual characteristics, such as being female, feeling lonely and worried, smoking cigarettes, drinking alcohol, and having fights at school, were associated with suicidal plans and behaviors regardless of the residential places. LIMITATIONS: A number of important variables have not been included in the questionnaire such as depression, family and parental support, household income, family constructs etc. CONCLUSION: Given the comparable patterns of risk between urban and rural adolescents and the relatively high rates of suicidal plans and attempts, similar mental health services and interventions are necessitated for both urban and rural areas.