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Yasmon A., Bela B., Ibrahim F., Syahruddin E.
41462004500;24723637900;54886001500;6507688750;
In vitro transcription of HIV-1 RNA for standard RNA
2011
Medical Journal of Indonesia
20
3
185
189
Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; Department of Pulmonology and Respiratory Medicine, Faculty of Medicine Universitas Indonesia/Persahabatan General Hospital, Jakarta, Indonesia
Yasmon, A., Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; Bela, B., Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; Ibrahim, F., Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; Syahruddin, E., Department of Pulmonology and Respiratory Medicine, Faculty of Medicine Universitas Indonesia/Persahabatan General Hospital, Jakarta, Indonesia
Background: The quantitative assays are important tests in the management of patients with HIV-1/AIDS. The important step in developing the assay is the availability of the standard HIV-1 RNA. For this purpose, we optimized in vitro HIV-1 RNA transcription to produce the standard HIV-1 RNA. Methods: The HIV-1 DNA was amplified from pNL43 by PCR using a primer pair that was specific for conserved region of HIV-1 Gag gene. The PCR product was further cloned into pBluescript II KS. The recombinant plasmid was restricted with EcoRI enzyme. Then, the linearized plasmid was used as template for RNA transcription. RT-PCR and PCR were performed simultaneously for confirmation of synthesized RNA fragment. Results: A 115 bp DNA of HIV-1 Gag gene has been cloned into pBluescript II SK with the exact true orientation. The reaction of the RNA transcription was also successfully performed. The RNA transcripts have been confirmed and showed the accuracy of the transcripts. Conclusion: we successfuly constructed the recombinant plasmid containing a conserved region of HIV-1 Gag gene, and the HIV-1 RNA has been transcribed in vitro as well. © 2011, Faculty of Medicine, Universitas Indonesia. All rights reserved.
HIV-1/AIDS; Quantitative assay; RNA transcription
DNA transcription; human; Human immunodeficiency virus 1; in vitro study; nonhuman; quantitative assay; recombinant plasmid; reverse transcription polymerase chain reaction; RNA transcription
Faculty of Medicine, Universitas Indonesia
08531773
Article
Q3