Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos

Ewart W Kuijk; Leonie du Puy; Helena T A van Tol; Henk P Haagsman; Ben Colenbrander; Bernard A J Roelen

doi:10.1186/1471-213X-7-58

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos

Ewart W Kuijk, Leonie du Puy, Helena T A van Tol, Henk P Haagsman, Ben Colenbrander, Bernard A J Roelen

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

BACKGROUND: In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction.

RESULTS: In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated.

CONCLUSION: Good reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either GAPDH or PGK1, together with ribosomal protein S18 (S18), and UBC is proposed as reference genes, but the use of B2M, BACT, or H2A is discouraged.

Original language	English
Pages (from-to)	58
Journal	BMC Developmental Biology
Volume	7
DOIs	https://doi.org/10.1186/1471-213X-7-58
Publication status	Published - 31 May 2007
Externally published	Yes

Keywords

Animals
Blastocyst/metabolism
Embryo Culture Techniques
Female
Gene Expression Profiling
Gene Expression Regulation, Developmental
Genes
Genes, Developmental
Oocytes/metabolism
RNA/analysis
Reference Standards
Reverse Transcriptase Polymerase Chain Reaction/standards
Swine

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10.1186/1471-213X-7-58

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title = "Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos",

abstract = "BACKGROUND: In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction.RESULTS: In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated.CONCLUSION: Good reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either GAPDH or PGK1, together with ribosomal protein S18 (S18), and UBC is proposed as reference genes, but the use of B2M, BACT, or H2A is discouraged.",

keywords = "Animals, Blastocyst/metabolism, Embryo Culture Techniques, Female, Gene Expression Profiling, Gene Expression Regulation, Developmental, Genes, Genes, Developmental, Oocytes/metabolism, RNA/analysis, Reference Standards, Reverse Transcriptase Polymerase Chain Reaction/standards, Swine",

author = "Kuijk, {Ewart W} and {du Puy}, Leonie and {van Tol}, {Helena T A} and Haagsman, {Henk P} and Ben Colenbrander and Roelen, {Bernard A J}",

year = "2007",

month = may,

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doi = "10.1186/1471-213X-7-58",

language = "English",

volume = "7",

pages = "58",

journal = "BMC Developmental Biology",

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T1 - Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos

AU - Kuijk, Ewart W

AU - du Puy, Leonie

AU - van Tol, Helena T A

AU - Haagsman, Henk P

AU - Colenbrander, Ben

AU - Roelen, Bernard A J

PY - 2007/5/31

Y1 - 2007/5/31

N2 - BACKGROUND: In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction.RESULTS: In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated.CONCLUSION: Good reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either GAPDH or PGK1, together with ribosomal protein S18 (S18), and UBC is proposed as reference genes, but the use of B2M, BACT, or H2A is discouraged.

AB - BACKGROUND: In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction.RESULTS: In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated.CONCLUSION: Good reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either GAPDH or PGK1, together with ribosomal protein S18 (S18), and UBC is proposed as reference genes, but the use of B2M, BACT, or H2A is discouraged.

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KW - Blastocyst/metabolism

KW - Embryo Culture Techniques

KW - Female

KW - Gene Expression Profiling

KW - Gene Expression Regulation, Developmental

KW - Genes

KW - Genes, Developmental

KW - Oocytes/metabolism

KW - RNA/analysis

KW - Reference Standards

KW - Reverse Transcriptase Polymerase Chain Reaction/standards

KW - Swine

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DO - 10.1186/1471-213X-7-58

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SN - 1471-213X

VL - 7

SP - 58

JO - BMC Developmental Biology

JF - BMC Developmental Biology

ER -

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos

Abstract

Keywords

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