Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR
| Autor: | Claudia Bagni, Brian T. Foley, Cecilia Graziosi, Antonio Mancarella, Giampietro Corradin, Elisa De Crignis, Giuseppe Pantaleo, Tilmann Achsel, Francesco A. Procopio |
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| Přispěvatelé: | Biochemistry |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Adult Male General Chemical Engineering 030106 microbiology Biology Gp41 Virus General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Open Reading Frames Young Adult SDG 3 - Good Health and Well-being Transcription (biology) Complementary DNA Humans RNA Antisense Gene General Immunology and Microbiology Reverse Transcriptase Polymerase Chain Reaction General Neuroscience virus diseases RNA Middle Aged Molecular biology Antisense RNA Open reading frame 030104 developmental biology HIV-1 |
| Zdroj: | Journal of Visualized Experiments (JoVE). MYJoVE Corporation ISSUE=153;ISSN=1940-087X;TITLE=Journal of Visualized Experiments (JoVE) |
| ISSN: | 1940-087X |
| Popis: | In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3' end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems. |
| Databáze: | OpenAIRE |
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