Development of a Competitive Quantitative PCR Strategy for Evaluating the Expression Stability of 18s rRNA during In Vitro Maturation of Buffalo (Bubalus bubalis) Follicular Oocytes

Abstract

The present work describes the development of a quantitative competitive PCR strategy for quantifying the relative abundance of 18s rRNA transcripts in buffalo oocytes during in vitro maturation (IVM). As a method, the competitive PCR overcomes some of the shortcomings of conventional reverse transcriptase polymerase chain reaction (RT-PCR) procedure making it a more authentic quantitative method. A composite primer based approach was used to generate the competitor cDNA to be used as external control. Validity of the method for its efficiency was demonstrated by quantitative analysis of the competition parameters. Using this method the relative abundance of buffalo oocyte 18s rRNA transcript over the period of IVM was found to vary within a narrow range of 0.93-1.06 folds which establishes the accuracy of the method and reflects the stability of its expression during IVM. This qualifies the use of this house keeping gene as a valid internal control in studies investigating the gene expression pattern in buffalo oocytes. The competitive PCR approach described in this study could be used for quantification of other transcripts from a limited number of oocytes where a conventional RT-PCR method is either difficult to use or multiplexing it with highly abundant house keeping genes is apparently problematic.

Expression Pattern of Apoptotic Genes in Vitrified-Thawed Bovine Oocytes

“…Using this method, 18S rRNA transcripts over the period of IVM including post-thaw vitrified oocytes varied within a narrow range of 0.5 and 0.8 cycles respectively, for oocytes derived from medium and small follicles, which establishes the accuracy of the method and reflects the stability of its expression during IVM. Our results support (Aswal et al 2007) with stable expression of 18S rRNA over the period of IVM of buffalo oocytes at five time points.…”

Identification and validation of reference genes for gene expression studies in water buffalo

“…Genes encoding for ribosomal proteins have been suggested as reference by Ayers et al (2007) for canine articular connective tissues, by Garcia-Crespo et al (2005) for ovine lymph node, by Goossens et al (2005) for bovine embryos, by Donaldson et al (2005) for mitogen-stimulated bovine cells, by Bionaz and Loor (2007) for bovine mammary gland and by JanovickGuretzy et al (2007) for bovine liver. Recently, Aswal et al (2007) evaluated with a semi-quantitative RT-PCR procedure the expression stability of 18s rRNA during the in vitro maturation of buffalo follicular oocytes. In this experimental system, the expression stability pattern of this gene was found superior than that of glyceraldehyde-3-phosphate dehydrogenase, a gene frequently used as endogenous control for quantitative RNA analysis (Aswal et al, 2008).…”

Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA Microarray

“…2006). Until recently, studies involving expression pattern of developmentally important genes in oocytes and embryos have not been investigated very widely in buffaloes and were confined to the use of quantitative real‐time polymerase chain reaction (PCR) using primers for specific transcripts designed based on bovine gene sequence (Aswal et al. 2007a,b).…”

“…2007a,b). Information on gene expression profiling in the course of IVM and IVF procedures in buffalo is crucial for optimization IVF and other assisted reproductive technologies (Aswal et al. 2007a,b).…”