In vitro transcription of HIV-1 RNA for standard RNA
Background: The quantitative assays are important tests in the management of patients with HIV-1/AIDS. The importantÂ step in developing the assay is the availability of the standard HIV-1 RNA. For this purpose, we optimized in vitro HIV-1Â 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 ofÂ HIV-1 Gag gene. The PCR product was further cloned into pBluescript II KS. The recombinant plasmid was restricted withÂ EcoRI enzyme. Then, the linearized plasmid was used as template for RNA transcription. RT-PCR and PCR were performedÂ 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. TheÂ reaction of the RNA transcription was also successfully performed. The RNA transcripts have been confirmed and showedÂ the accuracy of the transcripts.
Conclusion: We successfuly constructed the recombinant plasmid containing a conserved region of HIV-1 Gag gene, andÂ the HIV-1 RNA has been transcribed in vitro as well. (Med J Indones. 2011; 20:185-9)
Keywords: HIV-1/AIDS, Quantitative assay, RNA transcription
Copyright (c) 2011 Andi Yasmon, Budiman Bela, Fera Ibrahim, Elisna Syahruddin
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