Quantitative Reverse transcription-PCR Experiments Research Articles

Medicinal cannabis oil improves anxiety-like and depressive-like behaviors in CCS mice via the BDNF/TRPC6 signaling pathway

BackgroundPost-traumatic stress disorder (PTSD) refers to a chronic impairing psychiatric disorder occurring after exposure to the severe traumatic event. Studies have demonstrated that medicinal cannabis oil plays an important role in neuroprotection, but the mechanism by which it exerts anti-PTSD effects remains unclear. MethodsThe chronic complex stress (CCS) simulating the conditions of long voyage stress for 4 weeks was used to establish the PTSD mice model. After that, behavioral tests were used to evaluate PTSD-like behaviors in mice. Mouse brain tissue index was detected and hematoxylin-eosin staining was used to assess pathological changes in the hippocampus. The indicators of cell apoptosis and the BDNF/TRPC6 signaling activation in the mice hippocampus were detected by western blotting or real-time quantitative reverse transcription PCR experiments. ResultsWe established the PTSD mice model induced by CCS, which exhibited significant PTSD-like phenotypes, including increased anxiety-like and depression-like behaviors. Medicinal cannabis oil treatment significantly ameliorated PTSD-like behaviors and improved brain histomorphological abnormalities in CCS mice. Mechanistically, medicinal cannabis oil reduced CCS-induced cell apoptosis and enhanced the activation of BDNF/TRPC6 signaling pathway. ConclusionsWe constructed a PTSD model with CCS and medicinal cannabis oil that significantly improved anxiety-like and depressive-like behaviors in CCS mice, which may play an anti-PTSD role by stimulating the BDNF/TRPC6 signaling pathway.

Enhancer of zeste homolog 2 facilitates phenotypic transition of vascular smooth muscle cells leading to aortic aneurysm/dissection.

Thoracic aortic aneurysms (TAAs) are a major cause of death owing to weaker blood vessel walls and higher rupture rates in affected individuals. Vascular smooth muscle cells (VSMCs) are the predominant cell type within the aortic wall and their dysregulation may contribute to TAA progression. Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, is involved in several pathological processes; however, the biological functions and mechanisms underlying VSMC phenotype transition and vascular intimal hyperplasia remain unclear. The present study aimed to determine the involvement of EZH2 in mediating VSMC function in the development of TAAs. The expression of EZH2 was revealed to be elevated in patients with thoracic aortic dissection and TAA mouse model through western blotting and reverse transcription-quantitative PCR experiments. Subsequently, a mouse model was established using β-aminopropionitrile. In vitro, EdU labeling, Transwell assay, wound healing assay and hematoxylin-eosin staining revealed that knocking down the Ezh2 gene could reduce the proliferation, invasion, migration, and calcification of mouse primary aortic smooth muscle cells. Flow cytometry analysis found that EZH2 deficiency increased cell apoptosis. Depletion of Ezh2 in mouse primary aortic VSMCs promoted the transformation of VSMCs from a synthetic to a contractile phenotype. Using RNA-sequencing analysis, it was demonstrated that Ezh2 regulated a group of genes, including integrin β3 (Itgb3), which are critically involved in the extracellular matrix signaling pathway. qChIP found Ezh2 occupies the Itgb3 promoter, thereby suppressing the expression of Itgb3. Ezh2 promotes the invasion and calcification of VSMCs, and this promoting effect is partially reversed by co-knocking down Itgb3. In conclusion, the present study identified a previously unrecognized EZH2-ITGB3 regulatory axis and thus provides novel mechanistic insights into the pathophysiological function of EZH2. EZH2 may thus serve as a potential target for the management of TAAs.

Molecular characterization and functional analysis of a mollusk Rel homologous gene

Members of the nuclear factor-kappa B (NF-κB) family are crucial regulators of physiological processes such as apoptosis, inflammation, and the immune response, acting as vital transcription factors to perform their function. In this study, we identified a NF-κB homologous gene (CfRel1) in Zhikong scallops. The 3006-bp-long open reading frame encodes 1001 amino acids. The N-terminus of the CfRel1 protein harbors a conserved Rel homology domain (RHD) that contains a DNA-binding domain and a dimerization domain. According to the multiple sequence alignment results, both the DNA-binding and dimerization domains are highly conserved. Phylogenetic analysis indicated that CfRel1 is closely related to both the Dorsal protein of Pinctada fucata and the Rel2 protein of Crassostrea gigas. CfRel1 mRNA was expressed in all tissues tested in the quantitative reverse transcription PCR experiments, with hepatopancreatic tissue expressing the highest levels. Furthermore, after stimulation with lipopolysaccharide, peptidoglycan, or polyinosinic:polycytidylic acid, the mRNA expression level of CfRel1 was markedly increased. The co-immunoprecipitation test results showed that CfRel1 interacted with scallop IκB protein through its RHD DNA-binding domain, suggesting that IκB may regulate the activity of Rel1 by binding to this domain. Dual-luciferase reporter gene assays revealed that CfRel1 overexpression in HEK293T cells activated the activator protein 1 (AP-1), NF-κB, interferon (IFN)α, IFNβ, and IFNγ reporter genes, indicating the diverse functions of the protein. In summary, CfRel1 is capable of responding to attacks from pathogen-associated molecular patterns, participating in immune signaling, and activating NF-κB and IFN reporter genes. Our findings contribute to the advancement of invertebrate innate immunity theory, enrich the theory of comparative immunology, and serve as a reference for the future screening of disease-resistant strains in scallops.

Penumbra-targeted CircOGDH siRNA-loaded nanoparticles alleviate neuronal apoptosis in focal brain ischaemia

BackgroundNanoparticles (NPs) are a class of substances that can be loaded with therapeutic agents delivered to specific areas. In our earlier research, we identified a neuron-derived circular RNA (circRNA), circular...

Regulation of Multidrug Efflux Pumps by TetR Family Transcriptional Repressor Negatively Affects Secondary Metabolism in Streptomyces coelicolor A3(2).

Streptomyces spp. are well-known producers of bioactive secondary metabolites (SMs) that serve as pharmaceutical agents. In addition to their ability to produce SMs, Streptomyces spp. have evolved diverse membrane transport systems to protect cells against antibiotics produced by itself or other microorganisms. We previously screened mutants of Streptomyces coelicolor that show a phenotype of reduced undecylprodigiosin (RED) production in a combined-culture with Tsukamurella pulmonis. Here, we identified a point mutation, which reduced RED production, by performing genome resequencing and genetic complementation. We found that inactivation of the sco1718 gene encoding the TetR family transcriptional regulator (TFR) produced a deficient phenotype for several SMs in Streptomyces coelicolor A3(2). In the genome of S. coelicolor A3(2), two other sets of TFR and two-component ATP-binding cassette (ABC) transporter genes (sco4358-4360 and sco5384-5382) were found which had similar effects on the phenotype for both secondary metabolism and antibiotic resistance. An electrophoretic mobility shift assay and quantitative reverse transcription-PCR experiments demonstrated that TFRs repressed the expression of each adjacent two-component ABC transporter genes by binding to the operator sequence. Notably, the Δsco1718 mutant showed increased resistance to several antibiotics of other actinomycete origin. Our results imply the switching of cell metabolism to direct offense (antibiotic production) or defense (efflux pump activation) using costly and limited quantities of cell energy sources (e.g., ATP) in the soil ecosystem. IMPORTANCE The bacterial metabolic potential to synthesize diverse secondary metabolites in the environment has been revealed by recent (meta)genomics of both unculturable and culturable bacteria. These studies imply that bacteria are continuously exposed to harmful chemical compounds in the environment. Streptomyces spp. contain antibiotic efflux pumps and SM biosynthetic gene clusters. However, the mechanism by which soil bacteria, including Streptomyces, survive against toxic compounds in the environment remains unclear. Here, we identified three sets of TFR-ABC transporter genes in Streptomyces coelicolor A3(2). We found that each TFR controlled the expression of respective ABC transporter, and the expression of all ABC transporters negatively impacted SM production and increased antibiotic resistance. Notably, bioinformatic analysis indicated that these TFR-ABC transporter gene sets are highly conserved and widely distributed in the genome of Streptomyces species, indicating the importance of systematic regulation that directs antibiotic production and xenobiotic excretion.

Exosome-derived FGD5-AS1 promotes tumor-associated macrophage M2 polarization-mediated pancreatic cancer cell proliferation and metastasis

Inflammatory molecules and exosomes are crucial for signal transduction between tumor-associated macrophages and tumor cells. IL-6, a key inflammatory molecule secreted by M2 macrophages after polarization, can mediate malignant progression of pancreatic cancer (PC). However, the functions and mechanisms of IL-6 and tumor-derived exosomes in tumor-associated macrophages and PC remain unclear. Transcriptome chip and quantitative reverse transcription PCR experiments indicated that FGD5-AS1 induced IL-6 and high FGD5-AS1 expression correlated with the poor prognosis in PC patients. RNA pulldown, mass spectrometry, and dual luciferase reporter assays were used to identify the mechanism of exosomal FGD5-AS1 in promoting PC progression and M2 macrophage polarization. FGD5-AS1 exerted cancer-promoting functions when co-cultured with M2 macrophages. PC-derived exosomal FGD5-AS1 stimulated M2 macrophage polarization by activating STAT3/NF-κB pathway. FGD5-AS1 interacts with p300, resulting in STAT3 acetylation, thus promoting nuclear localization and transcriptional activity of STAT3/NF-κB. These data indicated that PC cells generate FGD5-AS1-rich exosomes, which cause M2 macrophage polarization to promote the malignant behaviors of PC cells. Targeting exosomal FGD5-AS1 may provide a potential diagnosis and treatment strategy for PC.

Validation of Circular RNAs Using RT-qPCR After Effective Removal of Linear RNAs by Ribonuclease R.

Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that have been shown to play a role in normal development, homeostasis, and disease, including cancer. CircRNAs are formed through a process called back-splicing, which results in a covalently closed loop with a nonlinear back-spliced junction (BSJ). In general, circRNA BSJs are predicted in RNA sequencing data using one of numerous circRNA detection algorithms. Selected circRNAs are then typically validated using an orthogonal method such as reverse transcription quantitative PCR (RT-qPCR) with circRNA-specific primers. However, linear transcripts originating from endogenous trans-splicing can lead to false-positive signals both in RNA sequencing and in RT-qPCR experiments. Therefore, it is essential to perform the RT-qPCR validation step only after linear RNAs have been degraded using an exonuclease such as ribonuclease R (RNase R). Several RNase R protocols are available for circRNA detection using RNA sequencing or RT-qPCR. These protocols-which vary in enzyme concentration, RNA input amount, incubation times, and cleanup steps-typically lack a detailed validated standard protocol and fail to provide a range of conditions that deliver accurate results. As such, some protocols use RNase R concentrations that are too high, resulting in partial degradation of the target circRNAs. Here, we describe an optimized workflow for circRNA validation, combining RNase R treatment and RT-qPCR. First, we outline the steps for circRNA primer design and qPCR assay validation. Then, we describe RNase R treatment of total RNA and, importantly, a subsequent essential buffer cleanup step. Lastly, we outline the steps to perform the RT-qPCR and discuss the downstream data analyses. © 2021 Wiley Periodicals LLC. Basic Protocol 1: CircRNA primer design and qPCR assay validation Basic Protocol 2: RNase R treatment, cleanup, and RT-qPCR.

Ubiquitin-conjugating enzyme 2b (HbUBC2b) is an appropriate reference gene for gene expression analysis by reverse transcription - quantitative PCR (RT-qPCR) in the rubber tree (Hevea brasiliensis) under mechanical wounding and methyl jasmonate treatment

The rubber tree (Hevea brasiliensis) is one of the most valuable members of the genus Hevea because it has high latex production and the latex exploited from the rubber tree is the primary source of natural rubber. Rubber trees are often damaged and exposed to pathogen attacks through wounds during and after tapping. Gene expression studies help us to gain insights into molecular mechanism underlying the responses of this plant species to stress signals such as wounding and pathogenicity. To ensure the reliability of gene expression measurements, quantitative procedures, particularly reverse transcription - quantitative PCR (RT-qPCR), require the normalization of the expression level of a gene of interest (target gene) to that of a stably expressed internal reference gene. Selecting and validating an appropriate reference gene is thus an essential step before conducting RT-qPCR experiments to look for differences in transcriptional expression of the target gene in H. brasiliensis under specific experimental conditions. Here, the transcriptional expression of ubiquitin-conjugating enzyme 2b (HbUBC2b) was measured by RT-qPCR in a set of 12 RNA samples derived from the bark of the rubber tree H. brasiliensis (clone RRIV 209) exposed to mechanical wounding, and stress signal molecules like methyl jasmonate (MeJA). With the aim of estimating PCR efficiency, and thus to include it in further analysis procedures, a standard curve assessment has been developed using HbUBC2b-specific primers and a 5-fold dilution of cDNA as template for qPCR reactions. The amplification efficiency (102,9%), as well as the R2 value (0,9992) are within the recommended range (90-110%). The HbUBC2b gene was shown to be stably expressed across control and treatment groups, and therefore could be used as a reference gene for quantitative gene expression normalization in H. brasiliensis (clone RRIV 209) under mechanical wounding or methyl jasmonate treatment.

Anti-CD206 antibody-conjugated Fe3O4-based PLGA nanoparticles selectively promote tumor-associated macrophages to polarize to the pro-inflammatory subtype.

M2 macrophages serve roles in inhibiting inflammation and promoting tumor development. Reversing tumor-associated macrophages (TAMs) from M2- to M1-type polarization may provide an important strategy for tumor immunotherapy. The present study aimed to enhance antitumor immunity by targeting the concentration of iron in macrophages. Fe3O4-based poly(lactic-co-glycolic) acid (PLGA) nanoparticles surface-modified with an anti-CD206 monoclonal antibody were prepared using the oil in water single-emulsion technique. Particle size was measured using a particle size analyzer, the ζ potential was determined using a ζ potential analyzer and the carrier rate of Fe3O4 was measured using an iron assay kit. The conjugation of anti-CD206, and the ability to target M2 macrophages were studied via immunofluorescence. Polarization indexes of the macrophages were detected using both western blotting and reverse transcription-quantitative PCR (RT-qPCR), and a mouse model with subcutaneous tumors was established to verify the antitumor effects of the nanoparticles in vivo. Nanoparticles had a mean diameter in the range of 260–295 nm, and the ζ potential values were between −19 and −33 mV. The Fe3O4 association efficiency ranged from 65–75%, whereas the anti-CD206 conjunction efficiency ranged from 65–70%. The immunofluorescence experiments were able to demonstrate the successful targeting of the M2 macrophages. The western blotting and RT-qPCR experiments identified that CD206-Fe3O4-PLGA and Fe3O4-PLGA promoted the expression of TNF-α, inducible nitric oxide synthase (iNOS) and IL-1β in the macrophages. The in vivo studies indicated that CD206-Fe3O4-PLGA nanoparticles were able to promote CD86 expression in TAMs, with CD86 being a specific marker of the M1 subtype. In summary, nanoparticles were characterized in the present study by their mean particle size, polydispersity index, ζ potential and morphology, as well as by their association with Fe3O4 and conjugation with the anti-CD206 monoclonal antibody. Collectively, the present results suggested that the nanoparticles were able to both target M2 macrophages and reverse the M2 polarization of the macrophages to the M1 phenotype via the release of coated iron-oxide particles.

Molecular and Phylogenetic Analyses of the Mediator Subunit Genes in Solanum lycopersicum.

The Mediator complex is a multi-subunit protein assembly that serves as a central scaffold to help regulate DNA-binding transcription factors (TFs) and RNA polymerase II (Pol II) activity controlled gene expression programmed in response to developmental or environmental factors. However, litter information about Mediator complex subunit (MED) genes in tomato is available, although it is an essential model plant. In this study, we retrieved 46 candidate SlMED genes from the genome of tomato, and a comprehensive analysis was conducted, including their phylogenetic relationship, chromosomal locations, gene structure, cis-regulatory elements prediction, as well as gene expression. The expression profiling of 46 SlMED genes was analyzed using publicly available RNA-seq data. Furthermore, we selected some SlMED genes to evaluate their expression patterns in various tissues and under different abiotic stress treatments by quantitative reverse transcription PCR experiments. This is the first detailed report to elucidate the molecular and phylogenetic features of the MED genes in tomato, and it provides valuable clues for further functional analysis in order to clarify the role of the SlMED genes in diverse plant growth, development and abiotic stress response.

Tissue-Specific Regulation of Na+ and K+ Transporters Explains Genotypic Differences in Salinity Stress Tolerance in Rice.

Rice (Oryza sativa) is a staple food that feeds more than half the world population. As rice is highly sensitive to soil salinity, current trends in soil salinization threaten global food security. To better understand the mechanistic basis of salinity tolerance in rice, three contrasting rice cultivars—Reiziq (tolerant), Doongara (moderately tolerant), and Koshihikari (sensitive)—were examined and the differences in operation of key ion transporters mediating ionic homeostasis in these genotypes were evaluated. Tolerant varieties had reduced Na+ translocation from roots to shoots. Electrophysiological and quantitative reverse transcription PCR experiments showed that tolerant genotypes possessed 2-fold higher net Na+ efflux capacity in the root elongation zone. Interestingly, this efflux was only partially mediated by the plasma membrane Na+/H+ antiporter (OsSOS1), suggesting involvement of some other exclusion mechanisms. No significant difference in Na+ exclusion from the mature root zones was found between cultivars, and the transcriptional changes in the salt overly sensitive signaling pathway genes in the elongation zone were not correlated with the genetic variability in salinity tolerance amongst genotypes. The most important hallmark of differential salinity tolerance was in the ability of the plant to retain K+ in both root zones. This trait was conferred by at least three complementary mechanisms: (1) its superior ability to activate H+-ATPase pump operation, both at transcriptional and functional levels; (2) reduced sensitivity of K+ efflux channels to reactive oxygen species; and (3) smaller upregulation in OsGORK and higher upregulation of OsAKT1 in tolerant cultivars in response to salt stress. These traits should be targeted in breeding programs aimed to improve salinity tolerance in commercial rice cultivars.

Identification of Endogenous Control miRNAs for RT-qPCR in T-Cell Acute Lymphoblastic Leukemia.

Optimal endogenous controls enable reliable normalization of microRNA (miRNA) expression in reverse-transcription quantitative PCR (RT-qPCR). This is particularly important when miRNAs are considered as candidate diagnostic or prognostic biomarkers. Universal endogenous controls are lacking, thus candidate normalizers must be evaluated individually for each experiment. Here we present a strategy that we applied to the identification of optimal control miRNAs for RT-qPCR profiling of miRNA expression in T-cell acute lymphoblastic leukemia (T-ALL) and in normal cells of T-lineage. First, using NormFinder for an iterative analysis of miRNA stability in our miRNA-seq data, we established the number of control miRNAs to be used in RT-qPCR. Then, we identified optimal control miRNAs by a comprehensive analysis of miRNA stability in miRNA-seq data and in RT-qPCR by analysis of RT-qPCR amplification efficiency and expression across a variety of T-lineage samples and T-ALL cell line culture conditions. We then showed the utility of the combination of three miRNAs as endogenous normalizers (hsa-miR-16-5p, hsa-miR-25-3p, and hsa-let-7a-5p). These miRNAs might serve as first-line candidate endogenous controls for RT-qPCR analysis of miRNAs in different types of T-lineage samples: T-ALL patient samples, T-ALL cell lines, normal immature thymocytes, and mature T-lymphocytes. The strategy we present is universal and can be transferred to other RT-qPCR experiments.

Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice.

The reliability of reverse transcription-quantitative PCR (RT-qPCR) results in gene expression studies depends on the approaches used to account for non-biological variations. In order to find a proper normalization strategy for the study of genes related to growth hormone signaling in skeletal muscle of growing mice, nine unrelated genes were evaluated as internal controls. According to the most used algorithms–geNorm, the Comparative ΔCq method, NormFinder and BestKeeper–GSK3B, YWHAZ, RPL13A and RN18S were found as the most stable. However, the relative expression levels of eight of the potential reference genes assessed decreased with age in cDNA samples obtained from the same amount of total RNA. In a different approach to analyze this apparent discrepancy, experiments were performed with cDNA obtained from equal amounts of purified mRNA. Since the decline was still observed, the hypothesis of an age-related change in mRNA to total RNA ratio that could account for the systematic decrease was rejected. Differences among experimental groups could be due to a substantial increase with age in highly expressed mRNAs, which would bias the quantitation of the remaining genes. Consequently, those reference genes reflecting this dilution effect, which would have been discarded considering their variable relative expression levels, arose as suitable internal controls.

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

The agronomical relevant tomato-Pseudomonas syringae pv. tomato pathosystem is widely used to explore and understand the underlying mechanisms of the plant immune response. Transcript abundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a common approach employed to investigate the possible role of a candidate gene in certain biological process under study. The accuracy of this technique relies heavily on the selection of adequate reference genes. Initially, genes derived from other techniques (such as Northern blots) were used as reference genes in RT-qPCR experiments, but recent studies in different systems suggest that many of these genes are not stably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq, provides an opportunity for the identification of transcriptionally stable genes that can be adopted as novel and robust reference genes. Here we take advantage of a large set of RNA-seq data originating from tomato leaves infiltrated with different immunity inducers and bacterial strains. We assessed and validated 9 genes that are much more stable than two traditional reference genes. Specifically, ARD2 and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RT-qPCR experiments involving this pathosystem.

Effects of growth conditions on archaellation and N-glycosylation in Methanococcus maripaludis.

In this study, the effects of growth conditions on archaellation in Methanococcus maripaludis were examined. Cells were grown in a variety of media, including complex, minimal and with formate as the electron donor, with different nitrogen sources, varied salinities and at a variety of growth temperatures. Of the conditions tested, Western blot results showed that major archaellin FlaB2 levels only varied detectably as a result of growth temperature. Whilst the amount of FlaB2 was similar for cells grown at < 35 °C, protein levels decreased at 38 °C and were barely detectable at 42 °C. Quantitative reverse transcription PCR experiments demonstrated that the flaB2 transcript levels were almost undetectable at 42 °C. Electron microscopy confirmed that the FlaB2 levels detected by Western blots corresponded to the state of archaellation, with cells grown at 42 °C being mostly non-archaellated. Unexpectedly, a lower apparent molecular mass for FlaB2 was observed in Western blots of cells grown at temperatures >38 °C, suggestive of a truncation in the attached N-linked tetrasaccharide at higher growth temperatures. MS analysis of archaella isolated from cells grown at 40 °C confirmed that FlaB2 was now decorated with a trisaccharide in which the third sugar was also lacking the attached threonine and acetamidino modifications found in the WT glycan.

Gene expression analysis in early embryos through reverse transcription quantitative PCR (RT-qPCR).

Real-time, reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive and reproducible technology for the analysis of gene expression patterns. Its ability to detect minute quantities of nucleic acid from multifarious sources makes it an ideal technique for embryonic transcript quantification. However, complex cellular diversity and active transcriptome dynamics in early embryos necessitate particular caution to avoid erroneous results. This chapter is intended to outline basic methodology to design and execute RT-qPCR experiments in pre-implantation embryos.

The Luc2 gene enhances reliability of bicistronic assays

Abstract Luciferases are prominent reporters in molecular and cellular biology investigations including miRNA target studies and the determination of Internal Ribosome Entry Site (IRES) activities in bicistronic assays. A majority of the current bicistronic vectors contain a firefly luciferase reporter as the second cistron. One reason for this is the presence of cryptic transcription start sites inside the luciferase gene. We present here an experimental evaluation of the cryptic transcription within the latest version of the firefly luciferase gene, luc2. Using flow cytometric analysis, we observed a negligible amount of cryptic transcriptional activity that was only slightly above the background of untransfected cells. Nevertheless, quantitative reverse transcription PCR experiments revealed a six-to-nine-fold gradual increase of transcription along the coding region of the gene. The level of cryptic transcription from the coding region of the improved luc2 firefly luciferase gene is significantly lower when compared to the luc+ gene. In summary, the luc2 better fulfills the requirements of bicistronic assays than the previous luc+ version. The observed low cryptic transcription activity in luc2 could be limiting only in cases where weak IRESs are studied.

Microarray analysis reveals genes and functional networks relevant to the predisposition to inverted teats in pigs1

The inverted teat defect is characterized by the failure of teats to protrude from the udder surface and has a negative effect on the economic efficiency of pig production. The inverted teat defect is influenced by genetic factors, but the number and identity of relevant genes are unknown. In this study, we compared the mRNA expression of teat tissues from unaffected pigs and affected pigs by using microarrays. Simultaneously, 24,123 probe sets were screened, of which some 15,000 had present calls and were analyzed for differential expression between mesenchymal and epithelial tissue of 3 categories of teats (i.e., normal teats of unaffected and affected animals, and inverted teats of the latter). Differential expression was more pronounced in epithelial than in mesenchymal tissue, and the comparisons among the 3 categories of teats showed that local processes at the side of the affected area as well as processes taking place at the level of the organ contribute to the development of inverted teats. Genes related to biofunctions of cell maintenance, proliferation, differentiation, and replacement; organismal, organ, and tissue development; genetic information and nucleic acid processing; and cell-to-cell signaling and interaction were differentially expressed, depending on the teat phenotype and the status of the animal as affected or unaffected. In particular, genes encoding members of canonical pathways of growth factor signaling were highlighted. Complementary to previous real-time quantitative reverse-transcription PCR experiments showing upregulation of growth factors (epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, platelet-derived growth factor, vascular endothelial growth factor) and their receptors in the inverted teat, here it is shown that the abundance of transcripts encoding subordinated proteins (acid phosphatase 1, soluble; activating transcription factor 2; casein kinase 2, α 1 polypeptide; casein kinase 2, α prime polypeptide; actinin, α 2; and Homo sapiens growth factor receptor-bound protein 2) within the growth factor signaling pathways are also affected. Tuning of the expression of genes of these pathways balances the differentiation and proliferation of epithelial and mesenchymal teat tissue and finally affects the shape and structure of the teats.

Stimulative effects of light and a temperature downshift on transcriptional expressions of developmentally regulated genes in the initial stages of fruiting-body formation of the basidiomycetous mushroomLentinula edodes

Quantitative reverse transcription PCR experiments were done to analyze the effects of light and a temperature downshift on transcriptional expressions of a variety of developmentally regulated genes in the initial stages of fruiting-body formation of Lentinula edodes wild-type strain FMC2. It was revealed that both proper light conditions and a temperature downshift are required for high levels of transcriptions of the genes and the formation of fruiting bodies. In L. edodes MIL-LEW-M13-1 strain, which has been reported previously to be capable of forming fruiting bodies without a low-temperature treatment, the expressions of the developmentally regulated genes were enhanced by light exposure.

Gene expression patterns during intramuscular fat development in cattle1

Deposition of intramuscular fat, or "marbling," in beef cattle contributes significantly to meat quality variables, including juiciness, flavor, and tenderness. The accumulation of intramuscular fat is largely influenced by the genetic background of cattle, as well as their age and nutrition. To identify genes that can be used as early biomarkers for the prediction of marbling capacity, we studied the muscle transcriptome of 2 cattle crossbreeds with contrasting intramuscular fat content. The transcriptomes of marbling LM tissue of heifers from Wagyu x Hereford (WxH; n = 6) and Piedmontese x Hereford (PxH; n = 7) crosses were profiled by using a combination of complementary DNA microarray and quantitative reverse transcription-PCR. Five biopsies of LM were taken from each animal at approximately 3, 7, 12, 20, and 25 mo from birth. Tissue was also collected from the LM of each animal at slaughter (approximately 30 mo). Microarray experiments, conducted on the first 3 biopsies of 2 animals from each crossbreed, identified 97 differentially expressed genes. The gene expression results indicated that the LM transcriptome of animals with high marbling potential (WxH) could be reliably distinguished from less marbled animals (PxH) when the animals were as young as 7 mo of age. At this early age, one cannot reliably determine meaningful differences in intramuscular fat deposition. We observed greater expression of a set of adipogenesis- and lipogenesis-related genes in the LM of young WxH animals compared with their PxH contemporaries. In contrast, genes highly expressed in PxH animals were associated with mitochondrial oxidative activity. Further quantitative reverse transcription-PCR experiments revealed that the messenger RNA of 6 of the lipogenesis-related genes also peaked at the age of 20 to 25 mo in WxH animals. The messenger RNA expression of ADIPOQ, SCD, and THRSP was highly correlated with intramuscular fat content of an individual in WxH animals. Our study provides clear evidence of early molecular changes associated with marbling and also identifies specific time frames when intramuscular fat development in cattle muscle can be detected by using gene expression. This information could be used by animal scientists to design optimal nutrition for high marbling potential. In addition, the genes found to be highly expressed during development of marbling could be used to develop genetic markers or biomarkers to assist with beef production strategies.