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 succesful 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.

Background: An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor γ coactivator 1α (PPARGC1A) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types. Results: The mRNA expression stability of 10 reference genes was determined. The expression of RPL13A and SDHA appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes (ACTB, TBP and TOP2B), the results not only showed that the mRNA expression of PPARGC1A was significantly higher in each of the longissimus dorsi muscle samples than in backfat (P < 0.05), but also that the expression was significantly higher in the most cranial of the three muscle samples (P < 0.05). Conclusion: This study provides a new set of reference genes (ACTB, TBP and TOP2B) suitable for normalization of real-time RT-PCR data of backfat and longissimus dorsi muscle in the pig. The obtained PPARGC1A expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass.

Background: Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses. Results: In the present study the gene transcription levels of 6 commonly used reference genes (ACTB, B2M, HPRT1, UBB, TUBA1 and RPL32) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, TUBA1, ACTB and UBB were found to be most stable in normal skin and B2M, ACTB and UBB in equine sarcoids. Conclusion: Based on these results, TUBA1, ACTB and UBB, respectively B2M, ACTB and UBB can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of UBB, ACTB and B2M can be recommended as a reliable and accurate normalisation factor.

Since the development of in vitro embryo production in cattle, different supplements have been added to culture media to support embryo development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birthweights and low viability in calves (Large Offspring Syndrome). Analysis of global gene expression in bovine embryos produced under different conditions can provide valuable information to optimize culture media for in vitro embryo production. We used RNA sequencing to examine the effect of in vitro embryo production, in either serum-containing or serum-free media, on the global gene expression pattern of individual bovine blastocysts. Compared to in vivo derived embryos, embryos produced in serum-containing medium had five times more differentially expressed genes than embryos produced in serum-free conditions (1109 vs. 207). Importantly, in vitro production in the presence of serum appeared to have a different impact on the embryos according to their sex, with male embryos having three times more genes differentially expressed than their female counterparts (1283 vs. 456). On the contrary, male and female embryos produced in serum-free conditions showed the same number (191 vs. 192) of genes expressed differentially; however, only 44 of those genes were common in both comparisons. The pathways affected by in vitro production differed depending on the type of supplementation. For example, embryos produced in serum-containing conditions had a lower expression of genes related to metabolism while embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism. Serum supplementation had a major impact on the gene expression pattern of embryos, with male embryos being the most affected. The transcriptome of embryos produced in serum-free conditions showed a greater resemblance to that of in vivo derived embryos, although genes involved in lipid metabolism were altered. Male embryos appeared to be most affected by suboptimal in vitro culture, i.e. in the presence of serum.

Background: Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation. First lineage differentiation starts at the blastocyst stage with the formation of the trophectoderm and the inner cell mass. The main objective of this study was the detection, identification and expression analysis of genes associated with blastocyst formation in order to help us better understand this process. This information could lead to improvements of in vitro embryo production procedures. Results: A subtractive cDNA library was constructed enriched for transcripts preferentially expressed at the blastocyst stage compared to the 2-cell and 8-cell stage. Sequence information was obtained for 65 randomly selected clones. The RNA expression levels of 12 candidate genes were determined throughout 3 stages of preimplantation embryo development (2-cell, 8-cell and blastocyst) and compared with the RNA expression levels of in vivo "golden standard" embryos using real-time PCR. The RNA expression profiles of 9 (75%) transcripts (KRT18, FN1, MYL6, ATP1B3, FTH1, HINT1, SLC25A5, ATP6V0B, RPL10) were in agreement with the subtractive cDNA cloning approach, whereas for the remaining 3 (25%) (ACTN1, COPE, EEF1A1) the RNA expression level was equal or even higher at the earlier developmental stages compared to the blastocyst stage. Moreover, significant differences in RNA expression levels were observed between in vitro and in vivo produced embryos. By immunofluorescent labelling, the protein expression of KRT18, FN1 and MYL6 was determined throughout bovine preimplantation embryo development and showed the same pattern as the RNA expression analyses. Conclusion: By subtractive cDNA cloning, candidate genes involved in blastocyst formation were identified. For several candidate genes, important differences in gene expression were observed between in vivo and in vitro produced embryos, reflecting the influence of the in vitro culture system on the embryonic gene expression. Both RNA and protein expression analysis demonstrated that KRT18, FN1 and MYL6 are differentially expressed during preimplantation embryo development and those genes can be considered as markers for bovine blastocyst formation.