Reverse Transcription qPCR Analysis Research Articles

P7C3 ameliorates barium chloride-induced skeletal muscle injury activating transcriptomic and epigenetic modulation of myogenic regulatory factors.

Skeletal muscle injury affects the quality of life in many pathologies, including volumetric muscle loss, contusion injury, and aging. We hypothesized that the nicotinamide phosphoribosyltransferase (Nampt) activator P7C3 improves muscle repair following injury. In the present study, we tested the effect of P7C3 (1-anilino-3-(3,6-dibromocarbazol-9-yl) propan-2-ol) on chemically induced muscle injury. Muscle injury was induced by injecting 50 µL 1.2% barium chloride (BaCl2) into the tibialis anterior (TA) muscle in C57Bl/6J wild-type male mice. Mice were then treated with either 10 mg/kg body weight of P7C3 or Vehicle intraperitoneally for 7 days and assessed for histological, biochemical, and molecular changes. In the present study, we show that the acute BaCl2-induced TA muscle injury was robust and the P7C3-treated mice displayed a significant increase in the total number of myonuclei and blood vessels, and decreased serum CK activity compared with vehicle-treated mice. The specificity of P7C3 was evaluated using Nampt+/- mice, which did not display any significant difference in muscle repair capacity among treated groups. RNA-sequencing analysis of the injured TA muscles displayed 368 and 212 genes to be exclusively expressed in P7C3 and Veh-treated mice, respectively. There was an increase in the expression of genes involved in cellular processes, inflammatory response, angiogenesis, and muscle development in P7C3 versus Veh-treated mice. Conversely, there is a decrease in muscle structure and function, myeloid cell differentiation, glutathione, and oxidation-reduction, drug metabolism, and circadian rhythm signaling pathways. Chromatin immunoprecipitation-quantitative polymerase chain reaction (qPCR) and reverse transcription-qPCR analyses identified increased Pax7, Myf5, MyoD, and Myogenin expression in P7C3-treated mice. Increased histone lysine (H3K) methylation and acetylation were observed in P7C3-treated mice, with significant upregulation in inflammatory markers. Moreover, P7C3 treatment significantly increased the myotube fusion index in the BaCl2-injured human skeletal muscle in vitro. P7C3 also inhibited the lipopolysaccharide-induced inflammatory response and mitochondrial membrane potential of RAW 264.7 macrophage cells. Overall, we demonstrate that P7C3 activates muscle stem cells and enhances muscle injury repair with increased angiogenesis.

Circ_0005397 enhances hepatocellular carcinoma progression through miR-1283/HEG1

Introduction and objectivesCircular RNA (circRNA) has been confirmed to be an important regulator for the progression of hepatocellular carcinoma (HCC). However, the role and regulatory mechanism of circ_0005397 in HCC are not completely clear. Patients and methodsFifty HCC patients were included in this study. Reverse transcription-qPCR analysis was used to appraise circ_0005397, microRNA (miR)-1283, HEG homolog 1 (HEG1) mRNA expression levels, while western blot was used to identify HEG1, PCNA, Bax and Bcl-2 protein expression levels. Furthermore, cell proliferation, apoptosis, migration, invasion and angiogenesis were judged through cell counting kit-8 assay, EdU assay, Caspase3 activity test, flow cytometry, transwell assay and tube formation experiment. Dual-luciferase reporter assay and RIP assay were used to verify the targeting relationship between miR-1283 and circ_0005397 or HEG1. Finally, the effect of circ_0005397 on HCC tumor development was detected by mice experiments in vivo. ResultsCirc_0005397 was highly expressed in HCC tissues and cells, in HCC cell lines. Circ_0005397 silencing inhibited proliferation, migration, invasion and angiogenesis, while induced apoptosis in HCC cells. Circ_0005397 could sponge miR-1283, and miR-1283 could target HEG1. MiR-1283 inhibitor incompletely counteracted the effect of si-circ_0005397 on HCC cell progression, while HEG1 overexpression partially overturned the effect of miR-1283 on HCC cell progression. Circ_0005397 regulated the expression of HEG1 through targeting miR-1283. Moreover, circ_0005397 silencing blocked the growth of HCC tumors in vivo. ConclusionsCirc_0005397 regulated HEG1 by targeting miR-1283, thereby promoting HCC development.

Coexisting mechanisms of bacterial community are changeable even under similar stable conditions in a chemostat culture

The coexisting mechanism of a synthetic bacterial community (SBC) was investigated to better understand how to manage microbial communities. The SBC was constructed with three kinds of phenol-utilizing bacteria, Pseudomonas sp. LAB-08, Comamonas testosteroni R2, and Cupriavidus sp. P-10, under chemostat conditions supplied with phenol as a sole carbon and energy source. Population densities of all strains were monitored by real-time quantitative PCR (qPCR) targeting the gene encoding the large subunit of phenol hydroxylase. Although the supply of phenol was stopped to allow perturbation in the SBC, all of the strains coexisted and the degradation of phenol was maintained for more than 800 days. The qPCR analyses showed that strains LAB-08 and R2 became dominant simultaneously, whereas strain P-10 was a minor population. This phenomenon was observed before and after the phenol-supply stoppage. The kinetic parameters for phenol of the SBC changed before and after the phenol-supply stoppage, which suggests a change in functional roles of strains in the SBC. Transcriptional levels of phenol hydroxylase and catechol dioxygenases of three strains were monitored by reverse-transcription qPCR (RT-qPCR). The RT-qPCR analyses revealed that all strains shared phenol and survived independently before the phenol-supply stoppage. After the stoppage, strain P-10 would incur the cost for degradation of phenol and catechol, whereas strains LAB-08 and R2 seemed to be cheaters using metabolites, indicating the development of the metabolic network. These results indicated that it is important for the management and redesign of microbial communities to understand the metabolism of bacterial communities.

Trefoil factor 1 and gastrokine 2 inhibit Helicobacter pylori-induced proliferation and inflammation in gastric cardia and distal carcinogenesis.

Helicobacter pylori (H. pylori) infection has been associated with non-cardia adenocarcinoma in the stomach, while its role in gastric cardia adenocarcinoma (GCA) remains controversial. In addition, the association between H. pylori and the protective factors trefoil factor 1 (TFF1) and gastrokine 2 (GKN2) in gastroesophageal adenocarcinomas has not been fully investigated. Therefore, the mRNA and protein expression levels of TFF1 and GKN2 in GCA and distal gastric adenocarcinoma (DGA) were analyzed using quantitative PCR (qPCR) and immunohistochemistry, and the association with H. pylori infection was investigated. In addition, the effects of TFF1 and GKN2 overexpression on H. pylori-induced cells were investigated using western blot and reverse transcription-qPCR analysis. The comparative analysis of 16S rRNA-positive mRNA expression between GCA and DGA showed no statistically significant difference. However, the rate of the H. pylori vacuolating toxin A (VacA) genotype was significantly higher in GCA (49.2%) compared with that in DGA (26.9%; P<0.05). H. pylori infection downregulated the mRNA and protein expression levels of TFF1 and GKN2 in gastric tumor tissues, and the mRNA expression level of TFF1 and GKN2 was also markedly decreased in vitro. Furthermore, the cell proliferation varied in H. pylori total protein treatment group with the different doses. Notably, treatment with 20 µg/ml H. pylori total protein for 24 h resulted in the highest cellular proliferation rate. In addition, TFF1 and GKN2 overexpression inversely inhibited H. pylori-induced cell proliferation and upregulated NF-κB, tumor necrosis factor-α, IL-1β, IL-2, IL-4 and IL-6. The results of the present study indicate that H. pylori, particularly the VacA+ strain, plays an important role in GCA pathogenesis in high-risk areas of China, while TFF1/GKN2 inhibits H. pylori-induced cell proliferation and inflammation in GCA and DGA.

Ethanol extract of Polyscias fruticosa leaves suppresses RANKL-mediated osteoclastogenesis in vitro and LPS-induced bone loss in vivo

BackgroundMany bone-related diseases such as osteoporosis and rheumatoid arthritis are commonly associated with the excessive activity of osteoclasts. Polyscias fruticosa has been used as traditional medicine for the treatment of ischemia and inflammation and also eaten as a salad. However, its effect on the bone related diseases has not been investigated yet. PurposeThis study aimed to investigate the effect of ethanol extract of P. fruticosa on RANKL-induced osteoclastogenesis in vitro and LPS-induced bone loss in mouse, and evaluate anti-osteoclastogenic activities of its major constituents. MethodsBMMs or RAW264.7 cells were treated with ethanol extract from P. fruticose leaves (EEPL), followed by an evaluation of cell viability, RANKL-induced osteoclast differentiation, actin-ring formation, and resorption pits activity. Effects of EEPL on RANKL-induced phosphorylation of MAPKs were evaluated by Western blotting. The expression levels of NFATc1 and c-Fos were evaluated by Western blotting or immunofluorescence assay. The expression levels of osteoclast-specific marker genes were evaluated by Western blotting and reverse transcription-qPCR analysis. A LPS-induced murine bone loss model was used to evaluate the protective effect of EEPL on inflammation-induced bone loss. HPLC analysis was performed to identify the major constituents of EEPL. ResultsEEPL significantly inhibited RANKL-induced osteoclast differentiation by decreasing the number of osteoclasts, osteoclast actin-ring formation, and bone resorption. EEPL suppressed RANKL-induced phosphorylation of p38 and JNK MAPKs, as well as the expression of c-Fos and NFATc1. EEPL decreased the expression levels of osteoclast marker genes, including MMP-9, TRAP and CtsK. Mice treated with EEPL significantly protected the mice from LPS-induced osteoclast formation and bone destruction as indicated by micro-CT and histological analysis of femurs. We also identified 3-O-[β-d-glucopyranosyl-(1→4)-β-d-glucuronopyranosyl] oleanolic acid 28-O-β-d-glucopyranosyl ester (1) and quercitrin (3) as the active constituents in EEPL for inhibiting RANKL-induced osteoclast differentiation. ConclusionThe results showed that EEPL exerted anti-osteoclastogenic activity in vitro and in vivo by inhibiting RANKL-induced osteoclast differentiation and function, and suggested that EEPL could have beneficial applications for preventing or inhibiting osteoclast-mediated bone diseases.

Stability, integrity, and recovery rate of cellular nucleic acids preserved in a new liquid-based cytology medium.

Liquid-based cytology (LBC) has replaced the conventional Papanicolaou test in cervical cancer screening. The cervical swab specimens collected in LBC media can also be used for additional analyses including high-risk HPV (HR-HPV) test, DNA methylation analysis, and HPV E6/E7 mRNA test. The stability, integrity, and recovery rate of cellular DNA and RNA after storage at different conditions were evaluated by a quantitative real-time PCR (qPCR) based HR-HPV test, reverse transcription qPCR (RT-qPCR), and agarose gel electrophoresis. Cervical swab specimens collected in a newly developed LBC medium, VersaMedium, and ThinPrep PreservCyt medium were processed on Hologic ThinPrep 5000 instrument. Cervical exfoliative cells fixed by VersaMedium exhibited good cellular morphology with intact membranes and delineated chromatin structures. Cellular DNA preserved in VersaMedium exhibited high level of stability at both room temperature and 4°C, and remained mostly intact at 4°C for up to 28 days. Cellular RNA preserved in VersaMedium maintained higher level of stability and integrity at 4°C than at room temperature. VersaMedium also showed no apparent adverse effect on the recovery rate of nucleic acids. In addition to maintaining cellular morphology, when stored at 4°C, VersaMedium preserves cellular nucleic acids and PreservCyt medium without noticeable adverse effects on the recovery rate during purification. Therefore, VersaMedium is an appropriate LBC medium for the collection and preservation of cervical swab specimens. And VersaMedium preserved cellular nucleic acids are of such high quality that they are suitable for HR-HPV qPCR test and RT-qPCR analyses.

Validation of Suitable Reference Genes for Quantitative Gene Expression Analysis in Panax ginseng.

Reverse transcription-qPCR (RT-qPCR) has become a popular method for gene expression studies. Its results require data normalization by housekeeping genes. No single gene is proved to be stably expressed under all experimental conditions. Therefore, systematic evaluation of reference genes is necessary. With the aim to identify optimum reference genes for RT-qPCR analysis of gene expression in different tissues of Panax ginseng and the seedlings grown under heat stress, we investigated the expression stability of eight candidate reference genes, including elongation factor 1-beta (EF1-β), elongation factor 1-gamma (EF1-γ), eukaryotic translation initiation factor 3G1 (IF3G1), eukaryotic translation initiation factor 3B (IF3B), actin (ACT), actin11 (ACT11), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and cyclophilin ABH-like protein (CYC), using four widely used computational programs: geNorm, Normfinder, BestKeeper, and the comparative ΔCt method. The results were then integrated using the web-based tool RefFinder. As a result, EF1-γ, IF3G1, and EF1-β were the three most stable genes in different tissues of P. ginseng, while IF3G1, ACT11, and GAPDH were the top three-ranked genes in seedlings treated with heat. Using three better reference genes alone or in combination as internal control, we examined the expression profiles of MAR, a multiple function-associated mRNA-like non-coding RNA (mlncRNA) in P. ginseng. Taken together, we recommended EF1-γ/IF3G1 and IF3G1/ACT11 as the suitable pair of reference genes for RT-qPCR analysis of gene expression in different tissues of P. ginseng and the seedlings grown under heat stress, respectively. The results serve as a foundation for future studies on P. ginseng functional genomics.

Transcriptional analysis of exopolysaccharides biosynthesis gene clusters in Lactobacillus plantarum.

Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and find applications also in non-dairy foods and in therapeutics. Recently, four clusters of genes (cps) associated with surface polysaccharide production have been identified in Lactobacillus plantarum WCFS1, a probiotic and food-associated lactobacillus. These clusters are involved in cell surface architecture and probably in release and/or exposure of immunomodulating bacterial molecules. Here we show a transcriptional analysis of these clusters. Indeed, RT-PCR experiments revealed that the cps loci are organized in five operons. Moreover, by reverse transcription-qPCR analysis performed on L. plantarum WCFS1 (wild type) and WCFS1-2 (ΔccpA), we demonstrated that expression of three cps clusters is under the control of the global regulator CcpA. These results, together with the identification of putative CcpA target sequences (catabolite responsive element CRE) in the regulatory region of four out of five transcriptional units, strongly suggest for the first time a role of the master regulator CcpA in EPS gene transcription among lactobacilli.

Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems.

Quantitative Polymerase Chain Reaction (qPCR) is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (Solanum lycopersicum L.) genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes (‘Santa Clara’, susceptible, and its near-isogenic line ‘LAM 157’, resistant); subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV) and its mock-inoculated control) and in two distinct times after inoculation (early and late). Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper). To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, TIP41 and EF1 were identified as the most stable genes for tomato-ToCMoV pathosystem, and TIP41 and EXP for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems.

Expression profile of circulating microRNAs as a promising fingerprint for cervical cancer diagnosis and monitoring.

Sensitive and specific biomarkers for the early detection of cervical cancer are urgently required to reduce the high morbidity and mortality of this disease. We previously demonstrated that circulating microRNAs (miRNAs) are correlated with certain types of human cancer. The aim of this study was to investigate the altered profile of serum miRNAs in cervical cancer patients in order to predict cervical cancer at a relative early stage. Serum samples were collected from 213 cervical cancer patients and 158 age- and ethnicity-matched controls. An initial screening of miRNA expression was performed by Solexa sequencing. Differential expression was validated using the stem-loop miRNA quantitative polymerase chain reaction (qPCR) assay in individual samples and the samples were arranged by two-phase selection and validation. The Solexa sequencing results revealed 12 markedly upregulated serum miRNAs in cervical cancer patients compared with controls. The reverse transcription-qPCR analysis identified a profile of 5 serum miRNAs (miR-21, -29a, -25, -200a and -486-5p) as a cervical cancer biomarker. The receiver operating characteristic curves indicated that a panel of 5 miRNAs constitutes a more sensitive and specific diagnostic test compared with any single miRNA-based assay, the squamous cell carcinoma antigen or the carbohydrate antigen 125. More importantly, miR-29a and miR-200a may indicate tumor histological grade and progression stage. Therefore, a 5-miRNA signature identified from genome-wide serum miRNA expression profiling may serve as a fingerprint for cervical cancer diagnosis.

Identification and expression analysis of ethylene biosynthesis and signaling genes provides insights into the early and late coffee cultivars ripening pathway

The plant hormone ethylene is involved in the regulation of a multitude of plant processes, ranging from seed germination to organ senescence. Ethylene induces fruit ripening in climacteric fruits, such as coffee, being directly involved in fruit ripening time and synchronization. Coffee early cultivars usually show a more uniform ripening process although little is known about the genetic factors that promote the earliness of ripening. Thus, this work aimed to characterize the putative members of the coffee (Coffea arabica) ethylene biosynthesis and signaling pathways, as well as to analyze the expression patterns of these members during fruit ripening of early (Catucaí 785-15) and late (Acauã) coffee cultivars. Reverse Transcription-qPCR analysis of the four biosynthesis genes (CaACS1-like; CaACO1-like; CaACO4-like e CaACO5-like) analyzed in this study showed that CaACO1-like and CaACO4-like displayed an expression pattern typically observed in climacteric fruits, being up-regulated during ripening. CaACS1-like gene expression was also up-regulated during fruit ripening of both cultivars, although in a much lesser extent when compared to the changes in CaACO1-like and CaACO4-like gene expression. CaACO5-like was only induced in raisin fruit and may be related to senescence processes. On the other hand, members of the ethylene signaling pathway (CaETR1-like, CaETR4-like, CaCTR2-like, CaEIN2-like, CaEIN3-like, CaERF1) showed slightly higher expression levels during the initial stages of development (green and yellow-green fruits), except for the ethylene receptors CaETR1-like and CaETR4-like, which were constitutively expressed and induced in cherry fruits, respectively. The higher ethylene production levels in Catucaí 785-15 fruits, indicated by the expression analysis of CaACO1-like and CaACO4-like, suggest that it promotes an enhanced CaETR4-like degradation, leading to an increase in ethylene sensitivity and consequently to an earliness in the ripening process of this cultivar. Ethylene production in Acauã fruits may not be sufficient to inactivate the CaETR4-like levels and thus ripening changes occur in a slower pace. Thus, the expression analysis of the ethylene biosynthesis and signaling genes suggests that ethylene is directly involved in the determination of the ripening time of coffee fruits, and CaACO1-like, CaACO4-like and CaETR4-like may display essential roles during coffee fruit ripening.

Genes for transport and metabolism of spermidine in Ruegeria pomeroyi DSS-3 and other marine bacteria

Spermidine, putrescine, and other polyamines are sources of labile carbon and nitrogen in marine environments, yet a thorough analysis of the functional genes encoding their transport and metabolism by marine bacteria has not been conducted. To begin this endeavor, we first identified genes that mediate spermidine processing in the model marine bacterium Ruegeria pomeroyi and then surveyed their abundance in other cultured and uncultured marine bacteria. R. pomeroyi cells were grown on spermidine under continuous culture conditions. Microarray-based transcriptional profiling and reverse transcription-qPCR analysis were used to identify the operon responsible for spermidine transport. Homologs from 2 of 3 known pathways for bacterial polyamine degradation were also identified in the R. pomeroyi genome and shown to be upregulated by spermidine. In an analysis of genome sequences of 109 cultured marine bacteria, homologs to polyamine transport and degradation genes were found in 55% of surveyed genomes. Likewise, analysis of marine meta- genomic data indicated that up to 32% of surface ocean bacterioplankton contain homologs for trans- port or degradation of polyamines. The degradation pathway genes puuB (γ-glutamyl-putrescine oxi- dase) and spuC (putrescine aminotransferase), which are part of the spermidine degradation pathway in R. pomeroyi, emerged as suitable targets for molecular-based studies of polyamine pro- cessing by marine bacterial communities. The frequency of genes encoding transport and catabolism of spermidine and related polyamines suggests an important role for these compounds in carbon and nitrogen budgets of marine bacterioplankton.