Dataset Information

Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos.

ABSTRACT: BACKGROUND: Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the successful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. RESULTS: In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA) were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst) in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. CONCLUSION: Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

SUBMITTER: Goossens K

PROVIDER: S-EPMC1315359 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos.

Goossens Karen K Van Poucke Mario M Van Soom Ann A Vandesompele Jo J Van Zeveren Alex A Peelman Luc J LJ

BMC developmental biology 20051203

<h4>Background</h4>Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the successful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a singl ...[more]

PMID: 16324220

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Project description:BackgroundCardiac arrest, and the associated arrest of blood circulation, immediately leads to permanent brain damage because of the exhaustion of oxygen, glucose and energy resources in the brain. Most hippocampal CA1 neurons die during the first week post the insult. Molecular data concerning the recovery after resuscitation are sparse and limited to the early time period. Expression analysis of marker genes via quantitative real-time RT-PCR enables to follow up the remodeling process. However, proper validation of the applied normalization strategy is a crucial prerequisite for reliable conclusions.Therefore, the present study aimed to determine the expression stability of ten commonly used reference genes (Actb, actin, beta; B2m, beta-2 microglobulin;CypA, cyclophilin A; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Hprt, hypoxanthine guanine phosphoribosyl transferase; Pgk1, phosphoglycerate kinase 1; Rpl13a, ribosomal protein L13A; Sdha, succinat dehydrogenase complex, subunit a, flavoprotein (Fp); Tbp, TATA box binding protein; Ywhaz, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) in the rat hippocampus four, seven and twenty-one days after cardiac arrest. Moreover, experimental groups treated with the anti-inflammatory and anti-apoptotic drug minocycline have been included in the study as well.ResultsThe microglial marker Mac-1, used as a target gene to validate the experimental model, was found to be upregulated about 10- to 20-fold after cardiac arrest. Expression stability of candidate reference genes was analyzed using geNorm and NormFinder software tools. Several of these genes behave rather stable. CypA and Pgk1 were identified by geNorm as the two most stable genes 4 and 21 days after asphyxial cardiac arrest, CypA and Gapdh at 7 days post treatment. B2m turned out to be the most variable candidate reference gene, being about 2-fold upregulated in the cardiac arrest treatment groups.ConclusionWe have validated endogenous control genes for qRT-PCR analysis of gene expression in rat hippocampus after resuscitation from cardiac arrest. For normalization purposes in gene profiling studies a combination of CypA and Pgk1 should be considered 4 and 21 days post injury, whereas CypA and Gapdh is the best combination at 7 days. CypA is most favorable if restriction to a single reference gene for all time points is required.

Dataset Information

Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos.

Publications

Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos.

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