Figure 1. qRT‐PCR set‐up and analysis. (a) The experimental set‐up of a qRT‐PCR reaction plate, demonstrating how three experimental conditions (EC1–EC3) can be compared on a single reaction plate. Rows A and B are amplified with beta‐actin‐specific primers, rows C and D with unspliced (US) RNA primers, rows E and F with multiple spliced (MS) RNA primers, and rows G and H with single‐spliced (SS) RNA primers (details are listed in Table 1). Columns 1–8 of rows A, C, E, and G are a standard dilution series of reference cDNA; column 9 of these rows is a no‐template control. Columns 10–12 have cDNA samples prepared without reverse transcriptase. Other wells have cDNA samples arrayed in triplicate (EC1–EC3) for the various conditions tested; (b) Amplification plot for the qRT‐PCR of a standard dilution series. Ten‐fold diluted templates are delayed in amplification. A threshold fluorescence value is defined, and the cycle at which the amplification profile exceeds this threshold is defined as the C _T value; (c) The C _T value has a log‐linear relationship with template abundance.

Figure 2. Analysis of HIV‐1 splice site usage. (a) Genome map of HIV‐1 indicating the positions of the open reading frames. The purple arrows indicate the binding sites for the primers used in the radiolabeled RT‐PCR. Both 4 kb and 2 kb RNAs are amplified with the same forward primer and a reverse primer unique to HIV‐1 RNA subclass (singly or multiply spliced) being analyzed. The red bars illustrate how viral exons are distributed on HIV‐1 RNAs; (b) Summary of the RNA major species in HIV‐1. The exons included in each RNA are listed with each included exon number separated by a solidus; (c,d) Cells were treated with scrambled siRNAs (scr), siRNA to luciferase (siluc), hnRNP A1 (siA1) or hnRNP A2 (siA2). Total RNA was extracted as detailed and analyzed for (c) levels of major HIV‐1 spliced forms (US, unspliced; SS, singly spliced; MS multiply spliced) or (d) splice site usage within specific HIV‐1 RNA classes following fractionation on polyacrylamide gels.