Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos.
The aim of the present study was to compare real-time polymerase chain reaction (PCR) and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited; for example, in studies of preimplantation embryonic development and to determine the variability of the real-time reverse transcription-PCR assay. The sensitivity, dynamic range and precision of both PCR systems were compared using a single mouse liver cDNA standard. The real-time system was 100-fold more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR extended for two orders of magnitude using a fixed end-point of 31 cycles. The percentage standard error of the mean based on 30 replicates was 0.14% of the threshold cycle (Ct) value for the real-time system and 6.8% for the end-point fluorescence intensity. The coefficients of variation (CV) for reverse transcription combined with real-time analysis and the complete gene expression protocol consisting of mRNA isolation, reverse transcription and real-time PCR analysis were 0.6% and 1.4% of the Ct values, respectively. The present paper details, for the first time, measurement of the biological variation of individual mammalian oocytes. The CV was 1.8% of the Ct value for expression analysis of six bovine oocytes. The results are discussed in relation to the analysis of gene expression in preimplantation embryo development.
Gál
Arpád Baji
AB
Agricultural Biotechnology Center, Institute of Animal Biology, Szent-Györgyi Albert u. 4, 2100 Gödöllo, Hungary. baji@abc.hu
Carnwath
Joseph Wallace
JW
Dinnyes
Andras
A
Herrmann
Doris
D
Niemann
Heiner
H
Wrenzycki
Christine
C
D000818:Animals; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics; D047108:Embryonic Development / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D008099:Liver / Q000378:metabolism; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D012333:RNA, Messenger