Journal: PLoS ONE
Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1
Figure Lengend Snippet: The full length bicistronic RNA is expressed from the dl VAR vectors. (A) Schematic representation of the experimental procedure to detect the full length bicistronic mRNA showing the primers and the size of the expected amplicons  . (B and D) HeLa cells were transfected with 200 ng of the dl HIV-1, dl ΔEMCV, or the different dl VAR plasmids. Total RNA was extracted from transfected cells and quantified. (B) Extracted RNA (3 µg) was used as template in a one-step RT-PCR designed to specifically detect the bicistronic RNAs (lanes 4, 6, 8, 10, 14, and 16). To assay for DNA contamination the same reaction was conducted in the absence of reverse transcriptase (lanes 5, 7, 9, 11, 13, 15, 17). In vitro transcribed dl HIV-1 IRES RNAs (lane 2) and water (lane 3) were included as RT-PCR controls. (C) Schematic representation of the experimental procedure to detect the RLuc-RNA and FLuc-RNA showing the primers and the size of the expected amplicons (D) Total RNA (200 ng) extracted from transfected HeLa cells was used as template in parallel RT-qPCR reactions designed to specifically detect the RLuc or FLuc containing RNAs. The RNA-FLuc concentration (pmol)/RNA-RLuc (pmol) ratio was calculated. Values are the means +/- SEM from three independent experiments (each RNA sample was amplified in three independent reactions).
Article Snippet: To generate plasmids without the SV40 mammalian promoter, the bicistronic vectors were digested with StuI and MluI (Fermentas), treated with the E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas).
Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Concentration Assay, Amplification