Relative Quantification Real-time PCR Research Articles

The viability of Leptospira is related to physicochemical properties of the surface water surrounding an agricultural area and HemO and LipL32 gene expression in response to iron in water

The pathogenic Leptospira can survive and contaminate surface water based on physicochemical factors. This study aimed to determine how the physicochemical properties of water sources influence the growth and effect of iron on the gene expression of Leptospira spp. P47. Surface water samples (n=55) were collected and used for Leptospira spp. P47 cultivation. Physicochemical factors, including iron, calcium, magnesium and pH, were analyzed. The association between Leptospira spp. P47 viability at days 5, 10 and 15 with the physicochemical factors were analyzed. In addition, this bacterium was cultured in six selected water samples. The effect of iron in water on HemO and LipL32 gene expression was determined by relative quantification real-time PCR. Leptospira viability at day 5 was not significantly correlated with physicochemical factors, while Leptospira viability at day 10 was associated with both pH and iron. The Leptospira viability rate at day 15 had a significantly positive association with pH and iron and a negative association with calcium. HemO expression was significantly increased, mostly in selected water samples and under iron-depleted conditions. Conversely, LipL32 expression was significantly decreased in all water samples. Physicochemical factors in natural surface waters are key factors for bacterial survival in the environment, which may increase the chance of Leptospira infection in humans.

Evaluation of the inhibitory effects of drugs on the growth of Babesia gibsoni using relative quantification real-time PCR.

To evaluate the inhibitory effects of drugs on the growth of Babesia gibsoni, relative quantification real-time PCR method was developed in this study. The 18S rRNA gene was used as a target gene for the 2-ΔΔCt method analysis. Additionally, chicken RNA was added to the parasitized blood before total RNA extraction. The chicken β-actin gene was selected as an internal control gene for the 2-ΔΔCt method analysis. The 100 µL parasitized blood samples with different percentages of parasitized erythrocytes (PPEs) (3%, 1.5%, 0.75%, 0.375% and 0.1875%) were prepared for relative quantification of B. gibsoni. Regression analysis results revealed significant linear relationships between the relative quantification value and parasitemia. 18S rRNA gene expression was significantly decreased after treatment with diminazene aceturate and artesunate in vitro drug sensitivity test. This result suggested that this relative quantification real-time PCR method can be used to evaluate the effects of drug inhibition.

Molecular effects of tobacco smoke and bile reflux in Barrett’s esophagus in vitro

Background: Chronic gastroesophageal-reflux disease creates an environment of high oxidative stress believed to contribute to the carcinogenic transformation from reflux esophagitis to the premalignant condition Barrett’s esophagus (BE). BE ultimately progresses to esophageal adenocarcinoma (EAC). The aim of this study was to confirm the molecular effects of tobacco smoke and bile reflux in the molecular carcinogenesis of Barrett’s adenocarcinoma. Methods: Tobacco smoke condensate (4,000 ng/mL and dilutions) and a standardized bile acid (0.4 mM) were used to determine effects on a human hTERT-immortalized non-neoplastic Barrett’s esophageal cell line (BarT) and a human esophageal squamous cell line (Het-1a). Fourteen clinically relevant genes were evaluated via relative quantification real-time PCR analysis. Results: There was a synergistic relationship regarding the effects of bile acids and smoke condensate on both the BarT and Het-1a cell lines. Genes for inflammatory mediators COX2, IL1β, IL6, and IL8 were significantly up-regulated by combination smoke and bile treatment in both cell lines, while genes vital for cellular protection such as superoxide dismutase genes, MUC family of genes, CDX2 and CYP3A4 had varying expression changes. Conclusions: Tobacco smoke may enhance harmful effects of bile-acid refluxate on esophageal mucosa and is a significant factor in EAC carcinogenesis.

The value of mRNA expression of S100A8 and S100A9 as blood-based biomarkers of inflammatory bowel disease

Background and study aimsNon-invasive biomarkers of inflammatory bowel diseases (IBD) are of critical importance. Here, we evaluated the S100A8 and S100A9 mRNA expression, as the heterodimers of calprotectin, in the blood leucocytes of IBD patients to find how their expression associates with the disease characteristics. Patients and methodsIn this cross-sectional study, 59 IBD patients and 30 healthy subjects were included. The flare and remission phases of disease were identified in 46 and 13 patients, respectively. Blood leucocytes were isolated, and the S100A8 and S100A9 mRNA expression were evaluated in the isolated leucocytes using relative quantification real-time PCR. ResultsThe mean S100A8 and S100A9 mRNA expression were significantly higher in IBD patients than in the controls (p = 0.03 and p = 0.02, respectively). The mean S100A8 and S100A9 mRNA expression were significantly higher in the flare phase of the disease compared with the remission phase (p = 0.01 and p = 0.007, respectively). S100A8 distinguished IBD patients from controls with the sensitivity and specificity of 73% and 64%, and flare phase of disease from remission with the sensitivity and specificity of 67% and 62%. On the other hand, S100A9 distinguished IBD patients from controls with the sensitivity and specificity of 81% and 70%, and flare phase of disease from remission with the sensitivity and specificity of 68% and 64%. ConclusionThe S100A8 and S100A9 mRNA are differentially expressed in blood leucocytes of IBD patients compared to healthy controls as well as active versus quiescent disease. Thus, they can be potentially used as a blood-based biomarker in the monitoring of IBD.

Identification of a carboxylesterase associated with resistance to naled in Bactrocera dorsalis (Hendel)

Compared to other organophosphate-resistant and -susceptible (S) lines of Bactrocera dorsalis, the carboxylesterase (CBE) BdE5 in the naled-resistant (nal-r) line has been found to possess remarkable quantitative elevation. Our study attempts to identify the role of BdE5 in naled resistance, and we discovered several points of interest. Firstly, activity staining on native PAGE revealed that the percentage of flies with intensive BdE5 bands in the nal-r line was substantially higher than in the S line, indicating that the BdE5 band correlates with naled susceptibility. Secondly, in vitro and in vivo inhibition assays showed that BdE5 was inhibited by naled in both lines; under diagnostic doses of naled, the overall extent of inhibition on CBEs was much greater in the S line than in the nal-r line. Thirdly, NanoLC-nanoESi-MS/MS analysis used the NCBI database to identify and annotate BdE5 as an esterase FE4-like (XP_011200445.1) in B. dorsalis. Fourthly, rapid amplification of cDNA ends was used to obtain the 2012-bp full-length BdE5 cDNA, which contained an open reading frame of 1770bp and encoded a putative protein of 590 amino acid residues. Phylogenetic analysis revealed that BdE5 is a secreted β-esterase (E clade) closely related to CG6414 (NP_570089), a CBE in Drosophila melanogaster. Finally, our relative quantification real-time PCR data showed a significant elevation in transcript levels of the BdE5 gene in nal-r line. Our results confirmed that BdE5 is correlated with naled resistance and provides further understanding about the identification and molecular characteristics of BdE5 in B. dorsalis.

Wine polyphenols exert antineoplasic effect on androgen resistant PC-3 cell line through the inhibition of the transcriptional activity of COX-2 promoter mediated by NF-κβ

ObjectiveMediterranean diet may play a role in the prevention of prostate cancer (PCa) development and progression. Cyclooxygenase-2 (COX-2) expression is associated with increased cellular proliferation, prevents apoptosis and favors tumor invasion. We intend to clarify whether resveratrol and other polyphenols effectively inhibit COX-2 activity and induce apoptosis in hormone-resistant PC-3 cell line. Material and methodPC-3 cells were cultured and treated with different concentrations of gallic acid, tannic acid, quercetin, and resveratrol in presence of phorbol myristate acetate (PMA; 50μg/ml) that induces COX-2 expression. Total RNA was extracted and COX-2 expression was analyzed by relative quantification real-time PCR (ΔΔCt method). COX-2 activity was determined by PGE-2 detection using ELISA. Caspase 3/7 luminescence assay was used to disclose apoptosis. Transitory transfection with short human COX-2 (phPES2 −327/+59) and p5xNF-κβ-Luc plasmids determined COX-2 promoter activity and specifically that dependant of NF-κβ. ResultsCOX-2 expression was not modified in media devoid of PMA. However, under PMA induction tannic acid (2.08±.21), gallic acid (2.46±.16), quercetin (1.78±.14) and resveratrol (1.15±.16) significantly inhibited COX-2 mRNA with respect to control (3.14±.07), what means a 34%, 23%, 46% and 61% reduction, respectively. The inhibition in the levels of PGE-2 followed a similar pattern. All compounds studied induced apoptosis at 48h, although at a different rate. PMA caused a rise in activity 7.4±.23 times phPES2 −327/+59 and 2.0±.1 times p5×NF-κβ-Luc at 6h compared to basal. Resveratrol suppressed these effects 17.1±.21 and 32.4±.18 times, respectively. Similarly, but to a lesser extent, the rest of evaluated polyphenols diminished PMA inductor effect on the activity of both promoters. ConclusionsPolyphenols inhibit transcriptional activity of COX-2 promoter mediated by NF-κβ. This effect could explain, at least in part, the induction of apoptosis in vitro by these substances in castration resistant PCa.

Los polifenoles del vino ejercen su efecto antineoplásico sobre la línea celular PC-3 andrógeno resistente a través de la inhibición de la actividad transcripcional del promotor de COX-2 mediada por NF-kβ

ObjectiveMediterranean diet may play a role in the prevention of prostate cancer (PCa) development and progression. Cyclooxygenase-2 (COX-2) expression is associated with increased cellular proliferation, prevents apoptosis and favors tumor invasion. We intend to clarify whether resveratrol and other polyphenols effectively inhibit COX-2 activity and induce apoptosis in hormone-resistant PC-3 cell line. Material and methodPC-3 cells were cultured and treated with different concentrations of gallic acid, tannic acid, quercetin, and resveratrol in presence of phorbol myristate acetate (PMA; 50μg/ml) that induces COX-2 expression. Total RNA was extracted and COX-2 expression was analyzed by relative quantification real-time PCR (ΔΔCt method). COX-2 activity was determined by PGE-2 detection using ELISA. Caspase 3/7 luminescence assay was used to disclose apoptosis. Transitory transfection with short human COX-2 (phPES2 -327/+59) and p5xNF-kβ-Luc plasmids determined COX-2 promoter activity and specifically that dependant of NF-kβ. ResultsCOX-2 expression was not modified in media devoid of PMA. However, under PMA induction tannic acid (2.08±.21), gallic acid (2.46±.16), quercetin (1.78±.14) and resveratrol (1.15±.16) significantly inhibited COX-2 mRNA with respect to control (3.14±.07), what means a 34%, 23%, 46% and 61% reduction, respectively. The inhibition in the levels of PGE-2 followed a similar pattern. All compounds studied induced apoptosis at 48h, although at a different rate. PMA caused a rise in activity 7.4±.23 times phPES2 -327/+59 and 2.0±.1 times p5xNF-kβ-Luc at 6h compared to basal. Resveratrol suppressed these effects 17.1±.21 and 32.4±.18 times, respectively. Similarly, but to a lesser extent, the rest of evaluated polyphenols diminished PMA inductor effect on the activity of both promoters. ConclusionsPolyphenols inhibit transcriptional activity of COX-2 promoter mediated by NF-kβ. This effect could explain, at least in part, the induction of apoptosis in vitro by these substances in castration resistant PCa.

Sodium Tanshinone Iia Sulfonate Inhibits Porcine Reproductive and Respiratory Syndrome Virus via Suppressing N Gene Expression and Blocking Virus-Induced Apoptosis

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic loss in the swine industry. Currently, there is no effective way to prevent PRRSV infection. Sodium tanshinone IIA sulfonate (STS), a natural compound derived from Salvia miltiorrhiza, was shown to possess anti-PRRSV activity, but the underlying mechanisms remain unclear. The objective of this study was to investigate the effect of STS on PRRSV-induced cell apoptosis and PRRSV N protein expression pattern. Relative quantification real-time PCR was used to evaluate the inhibition of STS on N gene expression. Simultaneously indirect immunofluorescence assay (IFA) and western blot were used to assess the effect on N protein expression. Apoptosis was analysed using fluorescence microscope with an annexin V-EGFP kit. The effect of STS on caspase-3 cleaving was assessed by western blot. Our results showed that STS could inhibit viral N gene expression at both the messenger RNA stage and at the protein level in PRRSV-infected cells in a dose-dependent manner. In addition, STS could also rescue PRRSV-induced apoptosis. Our data suggest that STS may serve as a base compound for developing more effective drugs against PRRSV infection.

Molecular characterisation of two homoeologous elicitor-responsive lipin genes in cotton

The identification and molecular characterisation of two lipin-like gene copies (GhLIPN) in cotton, Gossypium hirsutum, an allotetraploid derived from two progenitor diploid Gossypium species, is described. Sequence analyses of the GhLIPN copies, designated GhLIPN-1 and -2, revealed that they contain 11 exons, separated by ten introns. They each have a 2,643bp open reading frame that encodes 880 aa proteins, and share a 97.7 and 95.5% sequence similarity at the translated nucleotide and amino acid level, respectively. The GhLIPN genes have a distinct domain architecture consisting of an archetypical N-terminal lipin domain, followed by a haloacid dehalogenase (HAD) domain towards the C-terminus. A Southern blot did not distinguish between the two gene copies, which suggests that they may be homoeologs rather than paralogs. GhLIPN-2 is more similar to a homoeologous sequence from G. raimondii, representing the ancestral D-genome, compared to GhLIPN-1 that matches G. herbaceum and that represents the A-genome. Our data indicate that GhLIPN-1 and GhLIPN-2 are homoeologs that derive from the A- and the D-diploid genomes, respectively. The promoter sequences of GhLIPN-1 and -2 differ by 56%, as a result of multiple indels. In silico analysis of the promoter regions revealed that both genes contain numerous putative defence-related and elicitor-responsive cis-elements that support a role for GhLIPN in defence responses. Relative quantification real-time PCR confirmed the up-regulation in response to a cell-wall-derived V. dahliae elicitor, which supported the association of GhLIPN with defence signalling. The results add a new dimension to the proposed roles of lipins in plants by suggesting that lipins may have a role in defence signalling.

Dynamic distribution and tissue tropism of classical swine fever virus in experimentally infected pigs

BackgroundClassical swine fever (CSF), caused by the Classical swine fever virus (CSFV), is an Office International des Epizooties (OIE) notifiable disease. However, we are far from fully understand the distribution, tissue tropism, pathogenesis, replication and excretion of CSFV in pigs. In this report, we investigated the dynamic distribution and tissue tropism of the virus in internal organs of the experimentally infected pigs using real-time RT-PCR and immunohistochemistry (IHC).ResultsA relative quantification real-time PCR was established and used to detect the virus load in internal organs of the experimentally infected pigs. The study revealed that the virus was detected in all 21 of the internal organs and blood collected from pigs at day 1 to day 8 post infections, and had an increasing virus load from day 1 to day 8 post infections. However, there was irregular distribution virus load in most internal organs over the first 2 days post infection. Blood, lymphoid tissue, pancreas and ileum usually contain the highest viral loads, while heart, duodenum and brain show relatively low viral loads.ConclusionsAll the data suggest that CSFV had an increasing virus load from day 1 to day 8 post infections in experimentally infected pigs detected by real-time RT-PCR, which was in consistent with the result of the IHC staining. The data also show that CSFV was likely to reproduce in blood, lymphoid tissue, pancreas and the ileum, while unlikely to replicate in the heart, duodenum and brain. The results provide a foundation for further clarification of the pathogenic mechanism of CSFV in internal organs, and indicate that blood, lymphoid tissue, pancreas and ileum may be preferred sites of acute infection.

Construction and characterization of a cDNA library from shark regenerated hepatic tissue

Sharks are a type of fish with a full cartilaginous skeleton and have big livers. To better understand liver regeneration in sharks and screening for the important genes participated in disease-defense, in this study, a cDNA library of regenerated liver tissues of shark, Chiloscyllium plagiosum, was constructed. A total of 2103 expressed sequence tags (ESTs), which represents 997 unique genes, were sequenced. Among these genes, 434 (43.53%) of them showed significant similarity (E-values < 10 −5) to the sequences in NCBI Nt database, 685 (68.71%) of these unique genes showed significant similarity (E-values < 10 −5) to the sequences in NCBI Nr database, and 662 (66.40%) of these unique genes showed significant similarity (E-values < 10 −5) to the Swiss-Prot database. Preliminary analysis of unique genes according to COG database showed that unigenes were further grouped into 21 functional categories including inorganic ion transport and metabolism, energy production and conversion, posttranslational modification, protein turnover and chaperones, general function prediction only, translation, and ribosomal structure and biogenesis. Several possible candidate genes involved in liver regeneration were selected to analyze their expression with relative quantification real-time PCR. This study may contribute to our better understanding of the molecular mechanism of regeneration in shark liver. Furthermore, the EST cataloguing and profiling of shark will be also benefited to the functional genomic research in this marine species.

P06 Hepatic lumican expression in paediatric non-alcoholic fatty liver disease

IntroductionLumican is a glycoprotein involved in collagen cross-linking and modulation of the innate immune system. Overexpression of lumican was recently described in a group of adult patients with histologically progressive...

Erratum to: Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR

This article unfortunately contained a mistake. The strain designations of two species, Prevotella brevis and Prevotella bryantii, were inadvertently reversed in Table 2. The names and sequences of the corresponding species-specific primers were not in error. The corrected table appears below.

Expression Levels of p53 Messenger RNA Detected by Real Time PCR in Tumor Tissue, Lymph Nodes and Peripheral Blood of Patients with Non-Small Cell Lung Cancer—New Perspectives for Clinicopathological Application

ABSTRACTThe importance of p53 mutations in the pathogenesis of lung cancer is well established, but it is still controversial whether the presence of p53 mutations or overexpression of P53 protein are of clinical significance. The aim of this study was to define the levels of expression of p53 mRNA in tumor, lymph nodes and peripheral blood of patients with non-small cell lung cancer, detected by relative quantification real-time PCR and to analyze the association with clinicopathological data. We applied real-time PCR to define the levels of expression of p53 mRNA in tumor, lymph nodes and blood of 25 patients with non-small cell lung cancer, which underwent an operation at the Department of Thoracic Surgery of the University Hospital for Pulmonary Diseases in Sofia. As a control group 25 healthy volunteers were used. The method of relative quantification was applied for the analysis. We detected a high expression of p53 mRNA in 90.9% of the tumors and in 73.7% of the lymph nodes. A trend for a correlation between the high levels of p53 expression in the tumor and lymph nodes was also established (p<0.19). We did not find any correlation between p53 expression in tumor tissue and peripheral blood.The expression analysis of p53 messenger RNA, detected by real-time PCR, can supplement the knowledge of p53 as a biomarker for lung cancer diagnosis and pathogenesis. The method is more sensitive than the currently used methods for p53 expression analysis and thus provides opportunities for a more accurate clinical application of molecular markers.

Another formula for calculating the gene change rate in real-time RT-PCR

Relative quantification real-time PCR has become a routine method in molecular biology research to study the small amount gene expression. There are a few mathematical models for relative quantification data analysis in real-time PCR, and the formula 2(-DDCT ) is one of the most frequently used method. In this paper, we are to present another equation which directly calculates the change rate (R (c)) of gene expression, and give a example to compare the application of this model with 2(-DDCT).

Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR

Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.