VALIDATION OF REFERENCE GENES FOR THE QUANTITATIVE GENE EXPRESSION STUDIES IN THE OVARIAN FOLLICLES OF SHEEP
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Date
2012-12
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Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P
Abstract
ABSTRACT :
To confirm if the poor development of oocytes from cultured preantral follicles (PFs’) of sheep was indeed due to the aberrant expression of developmentally important genes, it is necessary to study the expression patterns of these genes in the cultured PFs’. While absolute and relative qRT-PCR are the two related techniques, relative quantification employing reference genes is the widely used method. Reference genes are assumed to be stably expressed in all tissues and under varied experimental conditions. However, such an assumption was frequently unfitting and therefore it was repeatedly emphasized that the reference genes need be validated for different tissues and experimental conditions. Accordingly in the present study six of the commonly used reference genes viz., ACTB, GAPDH, HPRT1, 18S rRNA, PPIA and RPLPO were validated for the gene expression studies in in vivo and in vitro grown PFs’ of sheep. The expression pattern of these reference genes in the granulosa cells and oocytes was studied by qRT-PCR in different development stages of in vivo and in vitro grown ovarian follicles. Data was analysed by RefFinder which employs four different software viz., ΔCt, Best keeper, Norm Finder and geNORM simultaneously to rank the genes according to their stability of expression and also suggests comprehensive ranking based on the results obtained in the four different software together.
From the results it is concluded that for the gene expression studies in sheep (i) granulosa cells from in vivo grown follicles RPLPO, GAPDH and PPIA, (ii) oocytes from in vivo grown follicles PPIA, HPRT1, RPLPO, (iii) granulosa cells from in vitro grown follicles PPIA, 18S rRNA, RPLPO, (iv) oocytes from in vitro grown follicles GAPDH, HPRT1, PPIA and (v) ovarian follicles as a whole whether in vivo or in vitro grown GAPDH, PPIA and HPRT1 may be used as reference genes.
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Keywords
SHEEP; OVARIAN FOLLICLES;