253 DIFFERENTIAL GENE EXPRESSION IN IN VIVO-DERIVED v. IN VITRO-PRODUCED EQUINE BLASTOCYSTS AS DETERMINED BY RT-qPCR

2010 ◽  
Vol 22 (1) ◽  
pp. 284
Author(s):  
K. Smits ◽  
K. Goossens ◽  
A. Van Soom ◽  
L. Peelman
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Differential Gene Expression ◽  
Real Time Pcr ◽  
Calf Serum ◽  
Fatty Acid Binding ◽  
Expression Levels ◽  
Differential Gene

Although in vitro production of equine embryos has greatly evolved in recent years, there are still substantial differences between in vitro-produced and in vivo-derived equine embryos. Fundamental insight into these differences could lead to optimization of equine assisted reproductive techniques. Reverse transcription quantitative real-time PCR (RT-qPCR) is a highly specific and sensitive tool to compare mRNA expression levels of specific genes and was used in this study to determine differences in gene expression between equine in vivo and in vitro embryos. In vivo embryos (n = 8) were derived by uterine flushing of artificially inseminated mares at 7 days after ovulation. For the production of the in vitro embryos (n = 8), oocytes from slaughtered mares were matured in DMEM-F12-based medium (Galli et al. 2007 Anim. Reprod. Sci. 98, 39-55) in 5% CO2 in air (maturation rate: 57%), fertilized by intracytoplasmic sperm injection, and cultured in DMEM-F12 with 10% fetal calf serum in 5% CO2, 5% O2, and 90% N2 for 9.5 days (cleavage rate: 74%; blastocyst rate: 7%). RNA was extracted from single early to expanded blastocysts and amplified and converted into cDNA with the WT-Ovation RNA Amplification System (NuGEN, San Carlos, CA, USA). Based on the presumed gene functions and differential gene expression as determined in a previously performed suppression subtractive hybridization (SSH; Smits et al. 2009 Reprod. Dom. Anim. 44, 75), 5 genes [brain expressed X-linked 2 (BEX2), Mps one binder kinase activator-like 3 (MOBKL3), fatty acid binding protein 3 (FABP3), minichromosome maintenance complex component 7 (MCM7), and ornithine decarboxylase (ODC)] were selected for quantification by RT-qPCR with the KAPA SYBR® FAST qPCR Kit (Kapa Biosystems, Belgium) on the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Nazareth, Belgium). All data were normalized with previously determined stable reference genes (beta actin, ubiquitin C, ribosomal protein L32, and glyceraldehyde-3-phosphate dehydrogenase) and statistically analyzed by means of a Mann-Whitney test. The fact that all genes were expressed at greater levels in the in vivo-derived blastocysts than in the in vitro-produced blastocysts confirmed the results of the SSH. This difference was highly significant for MOBKL3, BEX2, and ODC (P < 0.005), significant for FABP3 (P < 0.05), and not significant for MCM7. These genes have already been shown to be important for embryonic cell survival (ODC), oocyte maturation and pre- implantation development (MOBKL3) in mice, regulation during embryonic development (BEX2) and fetal development (FABP3) in human, and genome replication in eukaryotes (MCM7) (Pendeville et al. 2001 Mol. Cell Biol. 21, 6549-6558; Han et al. 2005 Nucleic Acids Res. 33, 6555-6565). In conclusion, 4 genes (MOBKL3, BEX2, ODC, and FABP3) with greater expression levels in in vivo-derived equine blastocysts have been identified. Whether the up-regulation of these genes is important for normal embryonic differentiation in the horse embryo is currently under investigation.

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

2020 ◽  
pp. 15-34
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Tissue Regeneration ◽  
Real Time Pcr ◽  
Molecular Level ◽  
Northern Blotting ◽  
Tissue Constructs ◽  
Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cryptococcus Neoformans ◽  
Differential Gene Expression ◽  
Fluorescent Protein ◽  
Differential Gene ◽  
Green Fluorescent

179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191 ◽  
Author(s):  
J. Angulo ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Expression Levels ◽  
The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

Abstract 559: Elevated Level of sCD40L During Acute Coronary Syndrome Leads to Systemic MIP-2 Release

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Thomas Stadlbauer ◽  
Benedikt Molitor ◽  
Wilhelm Jochen ◽  
Petra Harwaldt ◽  
Meike Schaub ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Real Time ◽  
Differential Gene Expression ◽  
Real Time Pcr ◽  
Transcriptional Level ◽  
Coronary Syndrome ◽  
Proinflammatory Mediators ◽  
Differential Gene ◽  
Regulated Gene Expression

Purpose: Acute coronary syndromes (ACS) have been associated with sCD40 Ligand (sCD40L) release. Endothial cells are located at the interface between the blood and parenchymal cells and take active part in many physiological and pathologic processes, including inflammation. It has been suggested that CD40L is a key player in the pathogenesis of atherosclerosis and ACS, originating from endothelial cells. When sCD40L is present in human plasma during ACS, it is mainly derived from activated platelets. This may lead to activation of endothelial cells resulting in atherosclerosis and eventually atherothrombosis. Therefore, the aim of the present study was to investigate differential gene expression profiles of endothelial cells after stimulation with sCD40L. Methods: Human endothelial cells (HUVEC) were stimulated with sCD40L for 2 hours. Differential gene expression patterns were evaluated with Oligonucleotide microarray hybridization (UG-U133A, Affymetrix Inc, Santa Clara, CA). Significantly up-regulated gene expression was confirmed and quantitated by Real-time-PCR as well as ELISA. In serum from ACS patients expression of sCD40L was correlated with these up-regulated genes products. Results: Stimulation of HUVEC with sCD40L leads to a profound release of proinflammatory mediators like IL-8, ICAM1, ISGF3 as well as MCP-1 on a transcriptional level. In addition, gene expression profiling revealed a highly significant increase in expression of the macrophage inflammatory protein 2 (MIP-2), also known as GRO-beta or CXCL2. This was quantitated as approximately 30 fold up-regulation by real-time-PCR as well as in ELISA analysis (8 fold at 8 to 24 hours) derived from supernations. In clinical serum samples from ACS patients there was a highly significant correlation between sCD40L and MIP-2 levels (r = 0.88, p < 0.01) Conclusion: Proinflammatory mediators as well as MIP-2 play an important role in the chemoattraction of macrophages, which underlines the key role of CD40L as a link between thrombotic events, e.g ACS, and inflammatory processes, e.g. atherosclerosis. Therefore targeting MIP-2 up-regulation might be a potential approach to prevent the progression of atherosclerotic diseases as well as a new therapeutic approach to treat ACS.

scholarly journals Differential gene expression between in vivo and in vitro maturation: a comparative study with bovine oocytes derived from the same donor pool

2019 ◽  
Author(s):  
Luiz Sergio Almeida Camargo ◽  
Michele Munk ◽  
Jose Nelio Sales ◽  
Sabine Wohlres-Viana ◽  
Carolina Capobiango Romano Quintão ◽  
...  
Keyword(s):  
Gene Expression ◽  
Comparative Study ◽  
Differential Gene Expression ◽  
In Vitro Maturation ◽  
Donor Pool ◽  
Differential Gene ◽  
Bovine Oocytes

scholarly journals Hypoxia and its possible relationship with endometrial receptivity in adenomyosis: a preliminary study

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Song Guo ◽  
Di Zhang ◽  
Xiaowei Lu ◽  
Qian Zhang ◽  
Ruihuan Gu ◽  
...  
Keyword(s):  
Real Time ◽  
Western Blotting ◽  
Real Time Pcr ◽  
Underlying Mechanism ◽  
Endometrial Receptivity ◽  
Control Group ◽  
Expression Levels ◽  
Preliminary Study

Abstract Background Adenomyosis (AM) is an important cause of female infertility. However, the underlying mechanism remains unclear. This report describes a preliminary study of hypoxia and its possible association with endometrial receptivity in AM. Methods The study was divided into in vitro and in vivo experiments. In vitro, expression levels of the endometrial receptivity markers HOXA10 and HOXA11 in the implantation period were examined using real-time PCR and western blotting. Endometrial expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, and HIF-3α was determined using immunohistochemistry. In vivo, using an AM mouse model established by oral administration of tamoxifen, we inhibited expression of HIF-2α using an HIF-2α antagonist (PT2399; 30 mg/kg body weight, twice daily by oral gavage for 2 days) and then examined expression levels of Hoxa10 and Hoxa11 using real-time PCR and western blotting. Results Endometrial mRNA and protein expression levels of HOXA10 and HOXA11 were significantly lower in patients with AM than in control patients. Expression of HIF-2α was significantly higher in the AM group than in the control group, whereas that of HIF-1α and HIF-3α was equivalent in both groups. In vivo analysis showed that administration of the HIF-2α antagonist resulted in increased expression of Hoxa10 and Hoxa11 at both the mRNA and protein levels in AM model mice. Conclusions HIF-2α overexpression may be one reason for decreased endometrial receptivity in AM. The current findings provide insight into HIF-2α-mediated AM-related infertility and suggest that PT2399 has potential as a treatment for AM. Trial registration This trial was retrospectively registered.

260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
A. Baji Gal ◽  
S. Mamo ◽  
S. Bodo ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Standard Error ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Mouse Embryos ◽  
Cell Stage ◽  
The Mean

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P &lt; 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

Sign in / Sign up

Export Citation Format

Share Document