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.
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.
Aswal
A P S
AP
Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India.
Datta
T K
TK
Raghav
S
S
De
S
S
Yadav
P
P
Goswami
S L
SL
D000818:Animals; D002020:Buffaloes / Q000502:physiology*; D002478:Cells, Cultured; D018076:DNA, Complementary; D047108:Embryonic Development / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012337:RNA, Ribosomal, 18S / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D012337:RNA, Ribosomal, 18S