RT-PCR for Autol DC Therapy Figure 4. Panel A: CFSE-low cells expressed as a percentage of total PBMCs. Mature DCs (CD209: 96%; CD14: 0%; CD80: 100%; CD83: 91%; CD86: 100%; HLA-DR: 96%; and HLA-I: 100%) were electroporated with 4 HIV antigen-encoding RNAs (hatched bar) or eGFP (solid bar) were cultured with CFSE-labeled PBMCs for 6 days. Frequency of CD8+ CFSE-low were cells determined by flow cytometry. Panel B: CD28/CD45RA phenotype of CD8+ cells induced to proliferate (CFSE-low) by DC electroporated with 4 HIV antigen-encoding RNAs (left panel), as compared to the frequency of CD8+ CFSE-low cells induced by eGFP-RNA loaded control DC (right panel), as determined by flow cytometry. Panel C: Frequency of IFN-c positive cells within the CD8+ CFSE-low subset induced by 4 hr re-stimulation with DC expressing individual HIV antigen-encoding RNAs, or eGFP control RNA, as determined by intracellular staining and flow cytometry. The background response for single HIV RNA stimulators (1ug HIV RNA/106 DC) was calculated at 0.38% from GFP RNA-electroporated DC (1ug GFP RNA/106 DC) and is indicated by the horizontal dashed line. doi:10.1371/journal.pone.0001489.g004 design. The strategy used to design primers for PCR amplification, using Rev sequence as an example, is summarized in Figure 1. As shown a primer that is complimentary to the consensus B sequence was designed (Rev F 7830). Alternative primers which could also accommodate the frequently found mutations in this location were designed as well. These alternative primers contain compensatory sequence variations to accommodate the frequently found mutations in positions 7847 and 7848 (Rev F 7830.1 and Rev F 7830.2). To minimize the number of primers used, a mismatch at the 59 end of a primer sequence was tolerated since lowering the PCR amplification stringency could compensate for such mismatches. However, mismatches at the 39 end of a primer sequence were avoided To enable transcription of the PCR product in vitro, the products of the primary PCR reaction were modified to insert a T7 RNA polymerase binding site at the 59 end (Figure 1). Naturally occurring translation initiation codons for Gag, Vpr and Nef were captured during PCR amplification. However Rev mRNA is formed in a transplicing event and capture of a full length cDNA via PCR is not achievable. Only the second exon of Rev is amplified, so the addition of the initiator ATG codon for the Rev antigen in a nested round of PCR is required in order to enable translation initiation. The reverse primers contain a poly(T)64 tail which is transcribed into a poly(A)64 tail on the synthesized RNAs. (Figure 1). Individual primer sequences for the primary round of amplification are provided in Table 1. Formulation of primer groups Oligonucleotides (IDT) were reconstituted at a concentration of 100 mM. Primers were combined into groups to reduce the number of PCR reactions (the composition of primer groups is provided in Table 2. The final primer concentration in formulated stock solutions was 5 mM for PCR, and 20 mM for gene-specific reverse transcription. The amplification protocol was simplified by grouping primers according to their location. The number of amplification reactions for each HIV antigen was significantly reduced from the scenario where individual primer combinations would be used: 6 for Gag, 4 for Vpr, 3 for Rev, and 2 for Nef. Once primer mixes were made they were not further changed and the same formulations of primers were used to amplify various plasma materials. PLoS ONE | www.plosone.org 8 January 2008 | Issue 1 | e1489