Abstract
BackgroundIn 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.ResultsIn 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.ConclusionGood 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.
Highlights
In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation
RT-PCR For each developmental stage, porcine oocytes and embryos with good morphology [26] were collected from three independent in vitro cultures [see Additional file 1], and RNA was isolated separately from these biological replicates. β-2-microglobulin (B2M), beta-actin (BACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2α (H2A), phosphoglycerate kinase 1 (PGK1), 18S ribosomal RNA (S18), and ubiquitin (UBC) were chosen as candidate reference genes and used for RT-PCR on oocytes. These genes are regularly used to normalise mRNA transcript levels and at least five of the genes have previously been used as reference genes in early development (BACT [27], GAPDH [28], H2A [29], S18 and UBC [30])
Three technical replicates were run in all quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) experiments, and all samples for one gene product were run on one 96-well plate to minimize inter-experimental variation
Summary
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. At the diplotene stage of an oocyte's first meiotic prophase, numerous genes are transcribed and translated, resulting in storage of mRNA and proteins that support early embryonic development. Oocytes arrest at the metaphase of the second meiotic division, where transcription is halted and translation of mRNA is reduced [1]. The start of zygotic genome activation varies between 1- and 8-cell stage embryos, dependent on the species [4]
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