Quantification of housekeeping transcript levels during the development of bovine preimplantation embryos.
In mammals, the study of gene expression in the preimplantation embryo has been difficult because the standard procedures used to quantify mRNA generally require large amounts of starting material. The development of protocols using different quantitative strategies generally involving the polymerase chain reaction (PCR) has provided new tools for exploration of gene expression in preimplantation embryos. However, the use of an internal standard, often referred as a housekeeping gene, is essential to normalize the mRNA levels. RNA levels of eight housekeeping genes were quantified using real time PCR throughout the preimplantation period of the bovine embryo to find the most suitable gene to be used as standard. Histone H2a was the best internal standard because the transcript levels were constant across the preimplantation period. Linear amplification of antisense RNA using the T7 promotor for in vitro transcription of the entire RNA pool was evaluated as a suitable way to preamplify the starting material prior to quantification and was effective in providing accurate RNA abundance profiles throughout the preimplantation period. However, the amplification appears to be template dependent because the amplification factors were higher for some genes.
Robert
Claude
C
Department of Animal Sciences, Centre de Recherche en Biologie de la Reproduction, Laval University, Québec, Canada G1K 7P4.
McGraw
Serge
S
Massicotte
Lyne
L
Pravetoni
Marco
M
Gandolfi
Fulvio
F
Sirard
Marc-André
MA
D000199:Actins / Q000235:genetics; D000818:Animals; D016375:Antisense Elements (Genetics); D001755:Blastocyst / Q000378:metabolism / Q000502:physiology*; D002417:Cattle; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006657:Histones / Q000235:genetics; D034921:Lamin Type B / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism / Q000592:standards*; D012337:RNA, Ribosomal, 18S; D012342:RNA, Small Nuclear; D014158:Transcription, Genetic; D014404:Tubulin / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics
D000199:Actins; D016375:Antisense Elements (Genetics); D006657:Histones; D034921:Lamin Type B; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012342:RNA, Small Nuclear; D014404:Tubulin; C405484:U2 small nuclear RNA; D025801:Ubiquitin