Computational protocol: Quantitative profiling of the full APOBEC3 mRNA repertoire in lymphocytes and tissues: implications for HIV 1 restriction

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Protocol publication

[…] Primer pairs were designed to avoid inter-A3 identity and only amplify the intended A3 target (A, Supplementary Figure S2, and Supplementary Table S1). Primer pair specificity was verified by manually inspecting alignments of primers and A3 cDNA sequences, by using BLAST (version 2.2.20; http://blast.ncbi.nlm.nih.gov/) and Primer-BLAST software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/; default parameters except E set to 1) and, experimentally, by attempting to amplify 104 copies of every A3 control template with each set of A3 qPCR primers (B). Primers were additionally designed to have similar melting temperatures and were confirmed to have similar reaction efficiencies (C and see below). Furthermore, primers were designed such that they would amplify all described variants of each A3 mRNA (i.e. splice variants and SNPs; data not shown). Design was assisted by the Roche ProbeFinder software (version 2.43; http://qpcr.probefinder.com/roche3.html) and by Primer3 software (version 0.4.0; http://primer3.sourceforge.net/). […]

Pipeline specifications

Software tools Primer-BLAST, Primer3
Application qPCR
Organisms Human immunodeficiency virus 1, Homo sapiens
Chemicals Cytidine