Applied Cell Biology
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An in house qPCR System to Evaluate Transcripts Stimulated by an Immune Innate Agonist (CIGB2020) during the Covid19 Pandemic Scenario
Jorge Aguiar*, José Angel Silva, Omar Gell, Regla Estrada, Freya Freire, Chabeli Rodríguez, Eduardo Pentón, Gerardo Guillén and Julio Cesar Aguilar
Biomedical Research Division, Center for Genetic Engineering and Biotechnology (CIGB); Ave 31 between 158 and 190. Cubanacan, Playa, P.O. Box 6162, Havana 10600, Cuba
Abstract
Background: A low cost and simple real-time PCR method to quantify different transcripts in human samples is a need in most developing countries. A quantitative real-time PCR assay to evaluate the relative RNA expression stimulation of specific transcripts during the Pandemic COVID19 scenario was developed at the Center for Genetic Engineering and Biotechnology in Havana, using a commercial amplification system with standards and specific primers that are produced in house.
Methods: Oro-pharyngeal scrape samples from patients that were treated previously with an innate immune agonist, the CIGB2020, were employed to RNA purification, cDNA synthesis, and evaluation of different gene transcripts by the in house qPCR assay using different standard curves constructed with specific primers that amplify a pool of the own evaluated transcripts.
Results: Preliminary results showed a typical standard curve from the in house qPCR system developed. A typical standard curve during the evaluation of the TLR3 gene transcript was described as example, and was calculated automatically by plotting the Ct values against each standard of known concentration, and the linear regression of this curve was also calculated as: M=-3.31641, B=42.73084, R=0.9516. It was also observed the influence of the CIGB2020 on the stimulation of the three toll like receptors from the innate immune system.
Conclusions: This study demonstrated that the in house qPCR system is useful to quantify the relatively induction of the expression of several genes transcripts considered as immunological markers of the immune innate system.
