Validação de primers para análises quantitativas por PCR em tempo real dos genes FSHR, LHR e HAS2 em tecidos caprinos

Freitas, Jeferson Lucas SousaRomcy, Kalil Andrade MubaracViana, Ana Clara Negreiros Parente Capela SampaioFreitas, Vicente José de FigueirêdoTeixeira, Dárcio Ítalo AlvesMelo, Luciana Magalhães

This study aimed to validate primers to amplify FSHR, LHR and HAS2 genes for quantitative analysisin goat. Thus, total RNA samples were extracted from both goat and bovine cumulus cells and reversetranscripted to obtain cDNA. Specific primer pairs were designed using Primer-BLAST software. First, primerconcentrations were adjusted to achieve the lowest Ct (threshold cycle). Subsequently, standard curves wereconstructed by serial dilution of cDNA. After primers concentrations adjustment, all primer pairs producedspecific amplicons in the presence of the target DNA. The optimized concentration of primer pairs was 1.0 μMand 0.8 μM for primers HAS2-1 and HAS2-2, and 3.0 μM for FSHR and LHR pair of primers. The primerefficiencies accessed by standard curves were: 1.01 for HAS2-1; 1.15 for HAS2-2; 1.02 for FSHR and 0.98 forLHR. In conclusion, all primers designed were appropriate for quantitative gene expression analysis purposes.(AU)

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