title: Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT.
abstract: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive and accurate tool for quantitative estimation of gene transcription levels in preimplantation embryos. To control for possible experimental variations, gene expression data must be normalized using internal control genes commonly known as reference genes. However, the stability of reference genes can vary depending on the state of development and/or experimental conditions; hence the assessment of their stability is essential before initiating a gene expression analysis. In the present study, we used RT-qPCR to measure the transcript levels of 10 commonly used reference genes and analyzed their expression stability in bovine blastocysts produced by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). Using the geNorm program, we found the best combination of genes to normalize gene expression data in bovine embryos at the blastocyst stage produced by IVF (HMBS, SF3A1, and HPRT1), ICSI (H2A, HMBS, and GAPDH), SCNT (ACTB, SF3A1, and SDHA) and/or between blastocysts produced by these methods (GAPDH, HMBS and EEF1A2). We also demonstrated that not only the culture conditions may affect the expression patterns in bovine blastocysts but also the choice of embryo production method may have an important effect.
authors.affiliation: Laboratorio de Reproducción,Centro de Biotecnología de La Reproducción (CEBIOR),Facultad de Medicina,Universidad de La Frontera,Temuco,Chile.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Luchsinger
authors.affiliation: Laboratorio de Reproducción,Centro de Biotecnología de La Reproducción (CEBIOR),Facultad de Medicina,Universidad de La Frontera,Temuco,Chile.
authors.forename: María Elena
authors.initials: ME
authors.lastname: Arias
authors.affiliation: Laboratorio de Reproducción,Centro de Biotecnología de La Reproducción (CEBIOR),Facultad de Medicina,Universidad de La Frontera,Temuco,Chile.
authors.forename: Tamara
authors.initials: T
authors.lastname: Vargas
authors.affiliation: Laboratorio de Investigación en Biotecnología Animal (LINBA),Facultad de Medicina,Universidad de La Frontera,Temuco,Chile.
authors.forename: Marcos
authors.initials: M
authors.lastname: Paredes
authors.affiliation: Laboratorio de Reproducción,Centro de Biotecnología de La Reproducción (CEBIOR),Facultad de Medicina,Universidad de La Frontera,Temuco,Chile.
authors.forename: Raúl
authors.initials: R
authors.lastname: Sánchez
authors.affiliation: Laboratorio de Reproducción,Centro de Biotecnología de La Reproducción (CEBIOR),Facultad de Ciencias Agropecuarias y Forestales,Universidad de La Frontera,PO Box 58-D,Temuco,Chile.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Felmer
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002417:Cattle; D042963:Electron Transport Complex II / Q000235:genetics; D005307:Fertilization in Vitro; D020869:Gene Expression Profiling; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D053652:Nuclear Transfer Techniques; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D020554:Sperm Injections, Intracytoplasmic
chemical_list: D020648:Peptide Elongation Factor 1; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D042963:Electron Transport Complex II