Detection of Gene Expression in Trace Cells of Early Porcine Embryo by Pre-amplified Quantitative PCR
This study aimed to compare 3 quantitative polymerase chain reaction(qPCR)methods for detecting the dynamic expression of genes associated with pluripotency and histone acetylase modification in trace cells of porcine early embryo.Porcine parthenogenetic activated embryos at different stages(1-cell,2-cell,4-cell,8-cell,morula and blastocyst)were collected,and the ex-pression of genes associated with cell pluripotency and histone acetylase modification were detec-ted by conventional RT-qPCR,cDNA pre-amplified qPCR and sample direct pre-amplified qPCR.The results indicated that the pre-amplified qPCR exhibited a stable amplification curve,and the melt curve displayed a consistent single peak,the conventional RT-qPCR produced cyclic thresh-olds above 35 and multiple peaks in the melt curve.Notably,target gene expression was success-fully detected even after a 20 000-fold dilution of embryonic cells using pre-amplification,and gene expression at the single embryonic cells could be reliably assessed.The expression patterns of genes related to pluripotency and histone acetylase modification exhibited an initial increase fol-lowed by a decline across different stages of porcine parthenogenetic activated embryos,with the highest expression levels occurring at the genome activation stage.In conclusion,pre-amplified qPCR demonstrates superior sensitivity and accuracy,with a relatively simple operational proto-col and lower costs,making it a suitable approach for gene expression analysis in trace cells of embryo.This methodology has the potential to advance the understanding for the mechanisms underlying early embryonic development.
preamplificationqPCRporcine early embryotrace cellsgene expression
万方数据
维普
