Validação de primers para análises quantitativas por PCR em tempo real dos genes GJA1, GJA4, UXT e 18S em tecidos caprinos

Freitas, Jeferson Lucas SousaViana, Ana Clara Negreiros Parente Capela SampaioRomcy, Kalil Andrade MubaracFreitas, Vicente José de FigueirêdoTeixeira, Dárcio Ítalo AlvesMelo, Luciana Magalhães

This study aimed to validate primers to amplify GJA1, GJA4, UXT and 18S genes for quantitativeanalysis in goats. Thus, total RNA was extracted from goat brain samples and reverse transcription wasperformed. Primer pairs were designed using Primer-BLAST software. First, primer concentrations (0.3, 0.6,0.8, 1.0 μM) were adjusted to achieve the lowest Ct (threshold cycle) value in qPCR reactions. Subsequently,standard curves were constructed by serial dilution of cDNA. After primer concentrations adjustment, all primerpairs produced specific amplicons in the presence of the target DNA. The optimized concentration of primerpairs was 0.6 μM to amplify most of the genes. At the standard curves, the following primer efficiencies ofamplification were found: 0.92 for gb18S; 1.03 for gbUXT; 1.03 for gbGJA1; 1.05 for gGJA1; 2.27 for gbGJA4;1.83 for gGJA4. In conclusion, for quantitative gene expression analysis, only the primers designed for UXT andGJA1 were appropriate.(AU)

1. genes de referência
2. qPCR
3. Goat
4. Caprino
5. reference gene