Selection of reference genes for studies of porcine endometrial gene expression on gestational day 12

Comparing gene expression patterns in the endometrium on gestational day 12 (GD12) between Erhualian (ER) and Landrace × Large White (LL) pigs is helpful to understand the biological mechanisms of fecundity. Selecting genes that have stable expression levels as the internal standards in a comparative study is essential for identifying real gene-specific variation by quantitative RT-PCR (qRT-PCR). Five genes expressed in sow endometria on GD12 were evaluated for their suitability as internal control for relative quantification by qRT-PCR. These genes were beta-actin (ACTB), beta-2-microglobulin (B2M), phosphoglycerate kinase 1 (PGK1), RNA polymerase II polypeptide G (RPG), and ribosomal protein S20 (RPS20), which represent different functional classes. Our results indicated that ACTB, B2M, and PGK1 were not suitable as internal standards for normalization because of their huge variability between the two breeds. RPS20 and RPG were most stable, and the former is recommended to serve as the internal standard when the use of multiple housekeeping genes is unpractical.     

Selection of reference genes for RT-qPCR analysis in Trichogramma chilonis (Hymenoptera: Trichogrammatidae)

Trichogramma chilonis is an important natural enemy for control of various Lepidoperan crop pests. The biology of T. chilonis is well-studied, but the molecular mechanisms of this biology require further study. Screening suitable reference genes is a vital step for use of RT-qPCR to understand underlying molecular physiology. In the present study, nine candidate reference genes including elongation factor 2 (EF2), ribosomal proteins (RPS23, RPL13, and RPL44), malate dehydrogenase (MDH), eukaryotic translation initiation factor 3 subunit F (EIF3F), zinc finger protein 268 (ZFP268), muscle specific protein 20 (MP20), and ATP synthase subunit alpha (ATP5F1A) were evaluated at different conditions including development stage, diet, temperature, and insecticide treatments. Four common algorithms (the Delta Ct method, geNorm, BestKeeper, and NormFinder) and RefFinder were used to analyze gene expression stability. Our results indicated that two reference genes used for normalization were sufficient, and the optimal combinations were: RPS23 and EF2 for developmental stages, ZFP268 and EF2 for feeding with different diets, ZFP268 and RPL13 for temperature treatments, and EF2 and RPL44 for insecticide treatments. The results provide preliminary determination of suitable reference gene for standard RT-qPCR analyses in T. chilonis, which might establish the foundation for further molecular biology research.     

夜香树花期荧光定量PCR内参基因的筛选

为筛选夜香树（Cestrum nocturnum L.）香气释放、生物钟等相关基因表达研究适用的内参基因,本研究采用夜香树盛花期叶片和花为实验材料,利用同源克隆和RACE技术,获得了夜香树6种经典的内参基因序列,分别为：Actb7、EF-1A、GAPDH、TUA、TUB2、UBQ;采用荧光定量PCR方法对18s rRNA和这6个内参基因的表达模式进行了分析,并通过Bestkeeper、geNorm、NormFinder 3种程序分析了内参基因的稳定性。结果表明,在花中,Actb7表达最稳定;在叶片中,EF-1A和UBQ的表达比较稳定;在2种组织中,EF-1A的表达相对稳定。3组稳定性分析中,geNorm程序确定的最佳内参基因数目均为2,最佳内参基因组合均为Actb7/EF-1A。本研究通过对稳定内参基因的筛选,以期为准确检测夜香树盛花期花瓣节律运动、香气释放、生物钟变化等相关基因的表达研究奠定基础。     

Evaluation of reference genes for quantitative real-time PCR in the brain,pituitary, and gonads of songbirds

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.     

Selection of reference genes for Harmonia axyridis (Coleoptera: Coccinellidae) feeding on different diets

The selection of a stable reference gene is vital to gene expression studies and to improving the accuracy of RT-qPCR data. With the deep research on the artificial feed of the Harmonia axyridis, there is an increasing need to evaluate the effects of feed on insects at the molecular level. To identify a reference gene to assess the expression of related genes in Harmonia axyridis (Pallas), ensure the reliability of target gene expression analysis using real-time PCR. Especially for H. axyridis were fed Rhopalosiphum padi (L.) or an artificial diet. In this study, the expression profiles of nine candidate reference genes, including 28SrRNA, 18SrRNA, RPS23, EF1, Actin, ATPase, GAPDH, UBI, RPL13 from different H. axyridis tissues (head, thorax, wing, leg, ovary, and fat body) were investigated. The stability of the nine candidate genes was assessed using geNorm, NormFinder, BestKeeper and RefFinder software, and a comprehensive analysis showed that EF1 is a suitable reference gene for eating different diets of different organizations from H. axyridis. 28SrRNA, 18SrRNA, and RPS23 can also be used as reference genes, but Actin, ATPase, RPL13 are not suitable as an internal reference gene.     

Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber

Quantitative real-time polymerase chain reaction (QRT-PCR) has become one of the most widely used methods for gene expression analysis. However, the expression profile of a target gene may be misinterpreted due to unstable expression of the reference genes under different experimental conditions. Thus, a systematic evaluation of these reference genes is necessary before experiments are performed. In this study, 10 putative reference genes were chosen for identifying expression stability using geNorm, NormFinder, and BestKeeper statistical algorithms in 12 different cucumber sample pools, including those from different plant tissues and from plants treated with hormones and abiotic stresses. EF1α and UBI-ep exhibited the most stable expression across all of the tested cucumber samples. In different tissues, in addition to expression of EF1α and UBI-ep, the expression of TUA was also stable and was considered as an appropriate reference gene. Evaluation of samples treated with different hormones revealed that TUA and UBI-ep were the most stably expressed genes. However, for abiotic stress treatments, only EF1α showed a relatively stable expression level. In conclusion, TUA, UBI-ep, and EF1α will be particularly helpful for reliable QRT-PCR data normalization in these types of samples. This study also provides guidelines for selecting different reference genes under different conditions.     

Selection of suitable reference genes from bone cells in large gradient high magnetic field based on GeNorm algorithm

Studies of animals and humans subjected to spaceflight demonstrate that weightlessness negatively affects the mass and mechanical properties of bone tissue. Bone cells could sense and respond to the gravity unloading, and genes sensitive to gravity change were considered to play a critical role in the mechanotransduction of bone cells. To evaluate the fold-change of gene expression, appropriate reference genes should be identified because there is no housekeeping gene having stable expression in all experimental conditions. Consequently, expression stability of ten candidate housekeeping genes were examined in osteoblast-like MC3T3-E1, osteocyte-like MLO-Y4, and preosteoclast-like FLG29.1 cells under different apparent gravities (μg, 1 g, and 2 g) in the high-intensity gradient magnetic field produced by a superconducting magnet. The results showed that the relative expression of these ten candidate housekeeping genes was different in different bone cells; Moreover, the most suitable reference genes of the same cells in altered gravity conditions were also different from that in strong magnetic field. It demonstrated the importance of selecting suitable reference genes in experimental set-ups. Furthermore, it provides an alternative choice to the traditionally accepted housekeeping genes used so far about studies of gravitational biology and magneto biology.     

Studies on the innervation of the endometrium

Summary The innervation of the endometrium of rabbit, rat, mink, mongoose and pig has been investigated electron microscopically. Large bundles of nerve fibers can be observed in the connective tissue spaces within the basal layer of the endometrium. Unmyelinated nerve fibers enter the lamina functionalis, terminal nerve fibers penetrate the basal lamina and make contact with the glandular and the cavum epithelial cells. The terminal axons contain abundant synaptic vesicles, dense core vesicles and mitochondria. To date, no specialized presynaptic or postsynaptic membranes have been found.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Grants Ku 210/5 and Be 524/4).Dedicated to Prof. Dr. Drs. h.c. Wolfgang Bargmann on his 70th birthday in friendship and admiration.     

The influence of recombinant feline oviductin on different aspects of domestic cat (Felis catus) IVF and embryo quality

Oviductin is known to be a key player providing a convenient environment for the process of fertilization affecting this by direct interaction with oocytes and sperm. As in vitro embryo production in the context of assisted reproduction for endangered felids is still in the process of optimization, oviductin might be used to improve IVF results. Recombinant His-tagged feline oviductin was expressed by transformed Escherichia coli BL21DE3 cells. The protein was purified by immobilized metal ion affinity chromatography. The effect of the recombinant protein was characterized in three experiments: a hemizona assay for sperm binding analysis, the IVF outcome, and the relative mRNA expression levels in blastocysts after IVF. A significant higher number of bound sperm cells were found after incubation in oviductin. No significant effect on cleavage, morula, and blastocyst rates with or without oviductin incubation during IVF could be observed. However, the relative mRNA abundance of GJA1, a gene, whose expression level is known to be a marker of embryo quality, was significantly increased (P value less than 0.05) in blastocysts after oviductin treatment. In contrast to this, expression of OCT4, HSP70, DNMT1, DNMT3A, BAX, IGF1R, and GAPDH was not significantly affected. We assume that our recombinant oviductin in its current nonglycosylated form is able to enhance sperm binding. Despite of a missing significant effect on IVF outcome, embryo quality in terms of relative GJA1 expression is influenced positively. These promising results demonstrate the value of recombinant oviductin for the IVF in cats.     

Male reproductive success in the domestic cat (Felis catus L.): A case history

The domestic cat shows a great variability in different life history traits like its social organization, ranging from a solitary life to living in large social groups depending on environmental conditions. Until now, the mating system has not been shown to vary between populations and was described as promiscuous. Here we present data on the reproductive success of a male, which clearly show that monopolization of females by males is possible in this species.     

Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.     

Identification of suitable reference genes for gene expression studies of human serous ovarian cancer by real-time polymerase chain reaction

Quantitative real-time RT-PCR (RT-qPCR) has proven to be a valuable molecular technique in gene expression quantification. Target gene expression levels are usually normalized to a stably expressed reference gene simultaneously determined in the same sample. It is critical to select optimal reference genes to interpret data generated by RT-qPCR. However, no suitable reference genes have been identified in human ovarian cancer to date. In this study, 10 housekeeping genes, ACTB, ALAS1, GAPDH, GUSB, HPRT1, PBGD, PPIA, PUM1, RPL29, and TBP as well as 18S rRNA that were already used in various studies were analyzed to determine their applicability. Totally 20 serous ovarian cancer specimens and 20 normal ovarian epithelial tissue specimens were examined. All candidate reference genes showed significant differences in expression between malignant and nonmalignant groups except GUSB, PPIA, and TBP. The expression stability and suitability of the 11 genes were validated employing geNorm and NormFinder. GUSB, PPIA, and TBP were demonstrated as the most stable reference genes and thus could be used as reference genes for normalization in gene profiling studies of serous ovarian cancer, while the combination of two genes (GUSB and PPIA) or the all three genes should be recommended as a much more reliable normalization strategy.     

Validation of Zebrafish （Danio redo） Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR （qRT-PCR） is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The fl-actin, EFlot and Rpll3ot genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1 or, Rpll3α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

Apoptosis in the canine endometrium during the estrous cycle

Apoptotic cell death in the endometria of 58 female dogs in different stages of the estrous cycle was assessed (in formalin-fixed, paraffin-embedded sections) with both the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and immunohistochemical detection of caspase-3 activity. For both techniques, the apoptotic index was determined in the surface epithelium, stroma, crypts, and basal glands by counting the percentage of stained cells in a total of 500 cells in each category. In the surface epithelium and stroma, TUNEL- and caspase-3-positive cells were rare (apoptotic index<1) throughout the estrous cycle. However, caspase-3 detection showed a significant increase in the apoptotic index in the stroma during anestrus as well as an increase in the index in both the stroma and surface epithelium in late metestrus. The apoptotic index increased during late metestrus and anestrus in the crypts and basal glands; in the crypts, this increase was significant only when caspase-3 detection was used, whereas in basal glands, significant differences were found for both techniques. In conclusion, apoptosis was present in canine endometrial cells during the estrous cycle, but caspase-3 detection showed more significant differences than the TUNEL assay. Furthermore, a high apoptotic index (suggestive of endometrial desquamation) was not detected in the surface epithelium and there was no significant correlation between the apoptotic index in any cell group and serum progesterone concentrations.