Use of Chuk as an internal standard suitable for quantitative RT-PCR in mouse preimplantation embryos.
Analysis of gene expression changes during preimplantation development by quantitative reverse transcription-polymerase chain reaction (Q-PCR) requires appropriate internal standards. Ideally, such a gene should show a constant level of transcripts per embryo across all preimplantation stages from unfertilized eggs to blastocysts. By analysing the microarray-based gene expression profiles of preimplantation embryos, it was found that a conserved helix-loop-helix ubiquitous kinase gene (Chuk, also known as IkappaB kinase alpha, IKKalpha or IKK1) satisfied this criterion. To test the utility of this gene as an internal standard for Q-PCR, the expression levels of two known genes (Nalp5/Mater, Pou5f1/Oct3/Oct4) were normalized by Chuk and other housekeeping genes (Actb, Gapdh, Eef1a1, and H2afz) and demonstrated that the former was more consistent with the expression patterns obtained by a whole-mount in-situ hybridization than those reported previously with the latter. It is concluded that Chuk, unlike other commonly used normalization controls, is a reliable and suitable internal standard for measuring gene expression levels by Q-PCR in mouse oocytes and preimplantation embryos.
Falco
Geppino
G
Developmental Genomics and Ageing Section, Laboratory of Genetics, National Institute on Ageing, National Institutes of Health, Baltimore, MD 21224, USA.
Stanghellini
Ilaria
I
Ko
Minoru S H
MS
D000818:Animals; D000941:Antigens / Q000096:biosynthesis; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004527:Egg Proteins / Q000096:biosynthesis; D005260:Female; D015870:Gene Expression / Q000502:physiology*; D051550:I-kappa B Kinase / Q000032:analysis / Q000096:biosynthesis*; D017403:In Situ Hybridization; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D000941:Antigens; D017931:DNA Primers; D004527:Egg Proteins; C416503:Nalp5 protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase