Dataset Information

Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

ABSTRACT: The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS), NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

SUBMITTER: Lin P

PROVIDER: S-EPMC3634797 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

Lin Pengfei P Lan Xiangli X Chen Fenglei F Yang Yanzhou Y Jin Yaping Y Wang Aihua A

PloS one 20130424 4

The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several ...[more]

PMID: 23638092

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Project description:Rhizophora apiculata is a halophytic, small mangrove tree distributed along the coastal regions of the tropical and subtropical areas of the world. They are natural genetic reservoirs of salt adaptation genes and offer a unique system to explore adaptive mechanisms under salinity stress. However, there are no reliable studies available on selection and validation of reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in R. apiculata physiological tissues and in salt stress conditions. The selection of appropriate candidate reference gene for normalization of qRT-PCR data is a crucial step towards relative analysis of gene expression. In the current study, seven genes such as elongation factor 1? (EF1?), Ubiquitin (UBQ), ?-tubulin (?-TUB), Actin (ACT), Ribulose1,5-bisphosphate carboxylase/oxygenase (rbcL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S) were selected and analyzed for their expression stability. Physiological tissues such as leaf, root, stem, and flower along with salt stress leaf samples were used for selection of candidate reference genes. The high-quality expression data was obtained from biological replicates and further analyzed using five different programs such as geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder. All algorithms comprehensively ranked EF1? followed by ACT as the most stable candidate reference genes in R. apiculata physiological tissues. Moreover, ?-TUB and 18S were ranked as moderately stable candidate reference genes, while GAPDH and rbcL were least stable reference genes. Under salt stress, EF1? was comprehensively recommended top-ranked candidate reference gene followed by ACT and 18S. In order to validate the identified most stable candidate reference genes, EF1?, ACT, 18S and UBQ were used for relative gene expression level of sodium/proton antiporter (NHX) gene under salt stress. The expression level of NHX varied according to the internal control which showed the importance of selection of appropriate reference gene. Taken together, this is the first ever systematic attempt of selection and validation of reference gene for qRT-PCR in R. apiculata physiological tissues and in salt stress. This study would promote gene expression profiling of salt stress tolerance related genes in R. apiculata.

Dataset Information

Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

Publications

Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

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