Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes.
Reprod Domest Anim
; 52(5): 722-730, 2017 Oct.
Article
en En
| MEDLINE
| ID: mdl-28407308
ABSTRACT
To ensure accurate normalization and quantification of target RNA transcripts using reverse transcription quantitative polymerase chain reaction (RT-qPCR), most studies focus on the identification of stably expressed gene(s) as internal reference. However, RNA preparation methods could also be an important factor, especially for test samples of limited quantity (e.g. oocytes). In this study, we aimed to select appropriate reference gene(s), and evaluate the effect of RNA preparation methods on gene expression quantification in porcine oocytes and cumulus cells during in vitro maturation. Expression profiles of seven genes (GAPDH, 18S, YWHAG, BACT, RPL4, HPRT1 and PPIA) were examined, on RNA samples extracted from cumulus cells (RNeasy Kit) and oocytes (RNeasy Kit and Lysis Kit) during in vitro maturation, respectively. Interestingly, different RNA preparation methods were found to potentially affect the quantification of reference gene expression in pig oocytes cultured in vitro. After geNorm analyses, the most suitable genes for normalization were identified, GAPDH/18S for cumulus cells and YWHAG/BACT for oocytes, respectively. Thus, our results provide useful data and information on the selection of better reference genes and RNA preparation method for related functional studies.
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Oocitos
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Porcinos
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ARN
/
Expresión Génica
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Reprod Domest Anim
Asunto de la revista:
MEDICINA REPRODUTIVA
/
MEDICINA VETERINARIA
Año:
2017
Tipo del documento:
Article
País de afiliación:
China