SYBR Green-based Real-time RT-PCR Research Articles

The expression miRNA-195 in a sample of Iraqi breast cancer patients.

Breast carcinoma is the most prevalent cancer-related cause of death in women, and metastasis is the main factor in morbidity. The total number of new cases of cancer in Iraq during the year 2019 was 35,864. New non-invasive prognostic biomarkers are needed for the rapid recognition and differentiation between breast cancer (BC) stages for treatment choice improvement. MicroRNA (miRNA) are small, noncoding RNAs regulating gene expression and involve many cellular processes, including metastasis. Circulating miRNAs (detected in the blood) show considerable potential as biomarkers for helping diagnosis or tracking treatment efficacy. Materials and Methods: total RNA was extracted from serum from (n=50) patients and (n=26) healthy control to measure the MicroRNA 195 expression using SYBR green-based real-time RT-PCR technology. As a result, the expression levels miR-195 in breast cancer patients’ serum were significantly increased (up-regulated) compared to those in the normal adjacent serum. BC group showed a higher significant miR-195 expression (upregulation) when compared with those in the control group. While the highest expression of miR195 was recorded in stage II. Keywords: Breast Cancer, MicroRNA, miR-195, Stage, Iraq

Metabolomic signature and molecular profile of normal and degenerated human intervertebral disc cells

BACKGROUND CONTEXTIntervertebral disc degeneration (IVDD) is an incurable, specific treatment-orphan disease with an increasing burden worldwide. Although great efforts have been made to develop new regenerative therapies, their clinical success is limited. PURPOSECharacterize the metabolomic and gene expression changes underpinning human disc degeneration. This study also aimed to disclose new molecular targets for developing and optimizing novel biological approaches for IVDD. STUDY DESIGNIntervertebral disc cells were obtained from IVDD patients undergoing circumferential arthrodesis surgery or from healthy subjects. Mimicking the harmful microenvironment of degenerated discs, cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) were exposed to the proinflammatory cytokine IL-1β and the adipokine leptin. The metabolomic signature and molecular profile of human disc cells were unraveled for the first time. METHODSThe metabolomic and lipidomic profiles of IVDD and healthy disc cells were analyzed by high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Gene expression was investigated by SYBR green-based quantitative real-time RT-PCR. Altered metabolites and gene expression were documented. RESULTSLipidomic analysis revealed decreased levels of triacylglycerols (TG), diacylglycerol (DG), fatty acids (FA), phosphatidylcholine (PC), lysophosphatidylinositols (LPI) and sphingomyelin (SM), and increased levels of bile acids (BA) and ceramides, likely promoting disc cell metabolism changing from glycolysis to fatty acid oxidation and following cell death. The gene expression profile of disc cells suggests LCN2 and LEAP2/GHRL as promising molecular therapeutic targets for disc degeneration and demonstrates the expression of genes related to inflammation (NOS2, COX2, IL-6, IL-8, IL-1β, and TNF-α) or encoding adipokines (PGRN, NAMPT, NUCB2, SERPINE2, and RARRES2), matrix metalloproteinases (MMP9 and MMP13), and vascular adhesion molecules (VCAM1). CONCLUSIONSAltogether, the presented results disclose the NP and AF cell biology changes from healthy to degenerated discs, allowing the identification of promising molecular therapeutic targets for intervertebral disc degeneration. CLINICAL SIGNIFICANCEOur results are relevant to improving current biological-based strategies aiming to repair IVD by restoring cellular lipid metabolites as well as adipokines homeostasis. Ultimately, our results will be valuable for successful, long-lasting relief of painful IVDD.

Examining the miR-93-5p and miR-17-5p Expression in Plasma and Breast Cancer Tissue as Possible Markers in Breast Cancer Prognosis

Background and Aim MicroRNAs (miRNAs) are a class of small non-coding RNAs (17-25 nucleotides) that have been studied in many diseases. miRNAs studies in different cancers have shown that miRNAs may be considered oncogene or tumor suppressor. So far, many studies have shown that miR-17-5p and miR-93-5p are important regulatory molecules in some biological processes, such as cell proliferation, associated with cancer formation. This study aimed to investigate and compare the tissue and plasma expression levels of miR-17-5p and miR-93-5p in patients with ductal carcinoma breast cancer with the normal control group. Methods & Materials The total RNA (including miRNA) was extracted from breast and plasma tissue samples of cancerous and normal samples. The RNA concentration and purity were confirmed using optical absorbance measurements. cDNA was synthesized, and the expression levels of miR-17-5p and miR-93-5p were assessed semi-quantitatively by SYBR Green-based real-time RT-PCR assay in plasma and breast tissues of ductal carcinoma breast cancer compared with the control normal samples with SNORD47 as internal normalizer. Data were statistically evaluated using GraphPad Prism 8.0.2. Ethical Considerations This study was approved by the Ethics Committee of the institute (IRAN 52d/4922, 6.10.2016). All study individuals signed a consent form to use their clinical samples and personal data under the physician’s supervision. Results The expression level of miR-17-5p showed significantly higher expression in tissues and plasma of the cancer group compared with the control group (P<0.0001). It was also significantly associated with tumor stage and lymph node, and ER (estrogen receptor) and PR (progesterone receptor) status (P<0.0001). While decreased expression of miR-93-5p in plasma and tumor tissues was shown to be significantly associated with tumor stage and lymph node involvement (P<0.0001). Conclusion The data revealed that high expression of miR-17-5p and low expression of miR-93-5p in both plasma and breast tumor might be associated with poor prognosis in breast cancer. However, miR-17-5p, due to the greater change in expression and ease of plasma detection, may serve as a possible non-invasive biomarker for breast cancer’s poor prognosis. Further follow-up studies are required to confirm this finding.

One-step SYBR green-based real-time RT-PCR assay for detection of foot-and-mouth disease virus circulating in India.

Rapid, sensitive, and reliable laboratory detection of foot-and-mouth disease virus (FMDV) infection is essential for containing and controlling virus infection in any geographical area. In this report a SYBR green-based 3Dpol-specific one-step real-time RT-PCR (rRT-PCR) assay was developed for the pan-serotype detection of FMDV in India. The detection limit of the SYBR green-based rRT-PCR was 10-2 TCID50/50µl, which is 10 times more sensitive than the traditional agarose gel electrophoresis-based RT-multiplex PCR (RT-mPCR). The standard curve exhibited a linear range across 8-log10 units of viral RNA dilution. The reproducibility and specificity of this assay were reasonably high suggesting that the 3Dpol-specific SYBR green rRT-PCR could detect FMDV genome specifically and with little run-to-run variation. The new 3Dpol-specific SYBR green rRT-PCR assay was evaluated alongside the established RT-mPCR using the archived FMDV isolates and clinical field samples from suspected FMD outbreaks. A perfect concordance was observed between the new rRT-PCR and the traditional RT-mPCR on viral RNA in the archived FMDV cell culture isolates tested. Furthermore, 73% of FMDV-suspected clinical samples were detected positive through the 3Dpol-specific SYBR green rRT-PCR, while the detection rate through the traditional RT-mPCR was 57%. Therefore, the SYBR green-based 3Dpol-specific one-step rRT-PCR could be considered as a valuable assay with higher diagnostic sensitivity to complement the routine assays that are being used for FMD virus diagnosis in India.

A SYBR Green-based real-time RT-PCR assay to differentiate the H1N1 influenza virus lineages

The H1N1 subtype influenza viruses (H1N1) have been causing persistent epidemics in human, swine and poultry populations since 1918. This subtype has evolved into four relatively stable genetic lineages, including classical swine influenza virus lineage, seasonal human influenza virus lineage, avian influenza virus lineage and Eurasian avian-like swine influenza virus lineage. In this study, four pairs of primers, based on the relatively conserved HA nucleotide regions of each H1N1 genetic lineage, were designed to establish a SYBR Green-based real-time quantitative RT-PCR (qPCR) assay to differentiate between the H1N1 genetic lineages. The results of qPCR assay showed that the lineage-specific primers designed for each H1N1 lineage were intra-lineage-specific, without mismatch of inter-lineage or inter-subtype and there appeared specific amplification curves when the concentrations of H1N1 plasmids were greater than or equal to 1.0 × 101 copies/reaction. Thus, this qPCR assay can specifically differentiate between the four lineages of H1N1 with a good specificity and sensitivity, which would assist in recognizing the infection and epidemic status of different H1N1 genetic lineages.

Serum microRNA-486-5p expression in obese Egyptian children and its possible association with fatty liver

AimsSeveral microRNAs (miRNAs) are involved in regulating the process of adipogenesis. White adipose tissue is a major source for these miRNAs. We aimed to evaluate the expression of miR-486-5p in children with obesity and its possible association with nonalcoholic fatty liver disease (NAFLD). MethodThis case-control study included 100 obese and overweight children and 100 normal-weight children of matched age and sex. All children were subjected to anthropometric measurements and evaluation of miR-486-5p expression levels using the SYBR green-based real-time RT-PCR technique. ResultsObese children showed significantly up-regulated miR-486-5p gene expression (p value < 0.001) when compared to control group. MiR-486-5p gene expression showed significant positive correlation with weight (r = 0.924), BMI (r = 0.497), waist circumference (r = 0.387), fat mass (r = 0.361), LDL(r = 0.351), TG (r = 0.867), TC (r = 0.875) and presence of fatty liver (r = 0.760). The best cutoff value of miR-486-5p gene expression in the prediction of obesity was 0.44 with AUC 0.736 that has a sensitivity 60% and specificity 90%, ConclusionThe serum level of the miR-486-5p gene is up-regulated in obese and overweight children and might be an independent predictor for obesity and fatty liver susceptibility.

One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection.

Due to the coronavirus pandemic, identifying the infected individuals has become key to limiting its spread. Virus nucleic acid real-time RT-PCR testing has become the current standard diagnostic method but high demand could lead to shortages. Therefore, we propose a detection strategy using a one-step nested RT-PCR. The nucleotide region in the ORF1ab gene that has the greatest differences between the human coronavirus and the bat coronavirus was selected. Primers were designed after that sequence. All diagnostic primers are species-specific since the 3´ end of the sequence differs from that of other species. A primer set also creates a synthetic positive control. Amplified products were seen in a 2.5% agarose gel, as well as in an SYBR Green-Based Real-Time RT-PCR. Amplification was achieved for the positive control and specific regions in both techniques. This new technique is flexible and easy to implement. It does not require a real-time thermocycler and can be interpreted in agarose gels, as well as adapted to quantify the viral genome. It has the advantage that if the coronavirus mutates in one of the key amplification nucleotides, at least one pair can still amplify, thanks to the four diagnostic primers.

Reverse transcriptase loop-mediated isothermal amplification and reverse transcriptase recombinase amplification assays for rapid and sensitive detection of cardamom vein clearing virus.

In the present study, two isothermal molecular assays viz. reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and reverse transcriptase recombinase amplification (RT-RPA) were developed to detect the cardamom vein clearing virus (CdVCV) infecting cardamom. Assays were optimized for parameters like duration, temperature and concentration of magnesium sulfate, and betaine in the case of RT-LAMP and magnesium acetate in the case of RT-RPA. Detection limits of both assays were determined and compared with conventional RT-PCR and SYBR Green-based real-time RT-PCR. RT-LAMP was found 10,000 times additional sensitive than RT-PCR and one-tenth that of real-time RT-PCR. RT-RPA was found 1000 times additional sensitive than RT-PCR and one-hundredth that of real-time RT-PCR. Both assays were specific, rapid, and sensitive for detecting CdVCV. Compared to real-time RT-PCR, these assays are economical and can be employed in large scale screening of cardamom plants against CdVCV for the selection of virus-free plants.

Clinical value of serum microRNA-195 expression in invasive ductal carcinoma of the breast

BackgroundMicroRNAs (miR) are small molecules, with around 17–25 nucleotides in length. Dysregulation of miRNA expression can be used as a diagnostic biomarker of cancer because of their high stability in human serum. MiR-195 has been implicated as a diagnostic biomarker of breast cancer. Therefore, this study aimed to identify the expression levels of miR-195 and its diagnostic role in patients of breast cancer (BC). MethodsThe expression patterns of miR-195 in the serum was analysed from a total number of 100 female with invasive ductal carcinoma in addition to healthy control subjects. Isolation of total RNA was done, samples were measured using SYBR green-based real-time RT-PCR technology. ResultsCirculating levels of miR-195 were found to be significantly decreased in BC group compared to that of control group (p < 0.001), the serum miR-195 was associated with numerous clinicopathological considerations of breast cancer as serum miR-195 expression decreased significantly in the presence of nodal (p = 0.044) and distant metastasis (p = 0.049) in BC cases, moreover it showed significant negative correlation with tumour stage, grade and primary tumour extent (p < 0.001). Receiver operating characteristic (ROC) curve was conducted for discrimination between BC cases and control groups, miR-195 expression showed excellent AUCs (0.987), with the best cut off value of 0.996, sensitivity was 90%, specificity was 100%, PPV was 100%, NPV was 87% and accuracy was 94%. Regression analysis revealed that higher CA15-3 and lower miRNA195 were considered as independent predictors for BC diagnosis. ConclusionOur data showed that serum miR-195 expressions were down-regulated in breast cancer females; furthermore, this downregulation was associated with higher specificity in diagnosis BC cases and so it could be used as a non-invasive diagnostic marker in BC.

Study of serum microRNA-99a relative expression as a diagnostic and prognostic noninvasive biomarker of breast cancer in Egyptian females

BackgroundBreast cancer (BC) is the most significant cause of malignancy deaths among females in Egypt as well as in other countries. Despite the evident miR-99a deregulation in many malignancies, including breast cancer, its serum level and clinical significance in breast cancer are still vague. This study assessed the role of dysregulation of serum levels of miR-99a in the diagnosis and prognosis of breast cancer in Egyptian females. MethodsBlood samples were obtained from 60 breast cancer females diagnosed as invasive ductal carcinoma and 40 healthy females of matched age. Samples were evaluated for miR-99a relative expression using SYBR green-based real-time RT-PCR technology. Also, the association between serum miR-99a expression and CA 15–3, the known tumor marker of breast cancer, was investigated. ResultsThe serum expression level of MiR-99a was lower in the breast cancer group than that of the control group (P < 0.001). MiR-99a was 76.7% sensitive and 95% specific in the diagnosis of breast cancer and its level correlated with many clinical parameters of breast cancer, including primary tumor extent (P = 0.021), TNM stage (P < 0.001), and histological grade of cancer (P = 0.003). Regression analysis revealed that lower miRNA 99a expression was considered as an independent predictor for the higher BC tumor stage. ConclusionExpression of serum miR-99a was downregulated in breast cancer offering early diagnosis and indicating poor prognosis of cases.

Development of a SYBR green-based real-time RT-PCR assay for rapid detection of the emerging swine acute diarrhea syndrome coronavirus

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus which was associated with severe diarrhea disease in pigs. SADS-CoV was first detected and identified as the causative agent of a devastating swine disease outbreak in southern China in 2017. Routine monitoring and early detection of the source of infection is therefore integral to the prevention and control of SADS-CoV infection. In this study, a SYBR green-based real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique was established for rapid detection and monitoring of this emerging virus. Specific primers were designed based on the conserved region within the M gene of the viral genome. The lowest detection limit of the RT-qPCR assay was 10 copies/μL. This assay was specific and had no cross-reaction with other 11 swine viruses. The positive rate of 84 clinical samples for the SYBR green-based RT-qPCR and the conventional RT-PCR was 73.81% (62/84) and 53.57% (45/84), respectively. These results demonstrated that the SYBR green-based RT-qPCR technique was an effectively diagnostic method with higher sensitivity than probe-based RT-qPCR and gel-based RT-PCR for detection and epidemiological investigations of SADS-CoV.

Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay.

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

A correlation between diameter of femur head and foot length at different gestational age of human foetus by manual measurement and its clinical significance

One-step SYBR Green I-based real-time RT-PCR assay for rapid detection as well as quantitation of Japanese encephalitis virus (JEV) in acute-phase patient CSF samples by targeting the NS3 gene was developed. The assay developed in this study was found to be more sensitive as compared to conventional RT-PCR. The specificity of the reported assay system was established through melting curve analysis as well as by cross-reactivity studies with related members of Flavivirus. The applicability of Real-time PCR assay for clinical diagnosis was validated with 32 suspected acute-phase CSF samples of Gorakhpur epidemic, India, 2005. The improved sensitivity of real-time RT-PCR was reflected by picking up 10 additional samples with low copy number of template in comparison to conventional RT-PCR. The quantitation of the viral load in acute-phase CSF samples was done using a standard curve obtained by plotting cycle threshold (Ct) values versus copy numbers of the RNA template. This is the first report on the application of real-time RT-PCR for detection as well as quantitation of JEV from patient CSF samples. These findings demonstrate the potential clinical application of the reported assay as a sensitive diagnostic test for rapid and real-time detection and quantitation of JEV in acute-phase clinical samples.

Efflux Pump AdeABC Assessment in Acinetobacter baumannii Strains Isolated in a Teaching Hospital

Over the past twenty years the worldwide clinically impact of Acinetobacter baumannii (A. baumannii) demonstrated its etiopathogenetic relevance. During a previously retrospective study in a teaching hospital, between January 2011 and February 2015, we observed increasingly infections caused by A. baumannii associated with antibiotic multi-resistance. Tigecycline, the first member of the glycylcycline class, is an effective option for the treatment of such infections even if, due to its increased clinical use, tigecycline resistant isolates have recently emerged. In A. baumannii several mechanisms are associated with a tigecycline decrease susceptibility, among these, expression efflux pump AdeABC and the presence of insertion sequence (IS) in the adeRS operon.About that, we decided to analyze adeB and adeS genes in 24 MDR A. baumannii clinical isolates, selected on the different tigecycline phenotype. The study of adeB and adeS genes was performed by an in-housepolymerase chain reaction (PCR) and by Sanger sequencing method. According to literature adeB and adeS genes were detected in all MDR A. baumannii isolates tested. Therefore our attention has focused on two resistant tigecycline clinical strains (ACI 2313 and ACI 1213), with a MIC value >8. In particular the ACI 2313 strains, showed the presence of an IS in the adeS gene. Then, adeS sequence analysis identified ISAba1 insertion. Moreover, adeB gene expression was evaluated by an in-house SYBR Green I-based real-time RT-PCR. We found an over expression of adeB gene in ACI 2313 strain, according to IS presence on adeS gene, while the lack of adeB overexpression in ACI 1213, still resistant to tigecycline, could be due to different resistance mechanisms.

Co-circulation in a single biome of the Juquitiba and Araraquara hantavirus detected in human sera in a sub-tropical region of Brazil.

Hantaviruses is an emerging infectious disease. Although HCPS has been reported in several regions of Brazil, more cases of HCPS have recently been reported in Minas Gerais than in any other state. In 2009, we analyzed 27 samples presenting antibodies against hantaviruses. These samples originated from 688 symptomatic patients, as determined based on the Hemorrhagic Fever Protocol. A subsequent SYBR Green-based real-time RT-PCR demonstrated the presence of the virus in 22 of the samples. Among the RT-PCR-positive samples, 17 were analyzed using DNA sequencing; these sequences were compared with others deposited in GenBank and showed similarity with the Araraquara and Juquitiba virus clusters. This work describe the detection of Juquitiba virus, including three fatal cases, in Minas Gerais state, furthermore, showed that it is feasible to characterize the circulating strains using a small fragment of S segment. Finally, the results suggest the co-circulation of Araraquara and Juquitiba virus in a single biome in Minas Gerais state.

Duplex realtime RT-PCR assay for the detection of Potato spindle tuber viroid (PSTVd) along with ef 1-α gene of potato

SYBR green based realtime RT-PCR assay coupled with melt curve analysis was developed for the detection of Potato spindle tuber viroid (PSTVd) along with and without internal control from potato. The amplification of the specific targets was identified by their melting points viz., 85.93±0.22, 85.62±0.34 and 82.07±0.23 for primer pairs PSTVd-1F/PSTVd-1R, PSTVd-NFP/PSTVd-NRP and PSTVd-QFP1/PSTVd-QRP1, respectively. The realtime RT-PCR assay was 1×104 times more sensitive than RT-PCR assay and the assay could detect up to 20 copies of the target using serially diluted plasmid and up to 0.025 fg of total RNA from infected tissues diluted in healthy plant RNA. Duplex realtime RT-PCR assay was also standardized to detect PSTVd along with elongation factor 1- α (ef-1α) gene, a stable housekeeping gene of potato. Duplex realtime RT-PCR assay showed two melt peaks indicating the successful amplification both the PSTVd RNA and internal control RNA. The developed assays could consistently detect PSTVd and proved to be highly sensitive and rapid for the detection of PSTVd in post entry quarantine testing.

Serum microRNA-195 is down-regulated in breast cancer: a potential marker for the diagnosis of breast cancer.

MicroRNA-195 (miR-195) is a tumor suppressor that plays an important role in tumorigenesis. There are few studies on miR-195 expression in breast cancer patients and the results have been inconsistent; therefore, this study examined miR-195 expression in the serum of BC patients. Samples from 102 normal subjects and 210 subjects with BC who had detailed clinical follow-up information available were selected. An internal reference (miR-16) and serum miR-195 were amplified and quantitatively detected by SYBR green-based real-time RT-PCR. We analyzed the differences in miR-195 levels between BC and healthy cases and the relationships between the miR-195 level and TNM stage and other clinicopathological parameters. In addition, changes in miR-195 levels were examined for 21 BC cases using paired samples before and after neoadjuvant chemotherapy. miR-195 was downregulated in BC compared with control samples (P = 0.000, Mann-Whitney U test). The sensitivity and specificity of miR-195 in the diagnosis of BC were 69.0 and 89.2 %, respectively; whereas, the sensitivities of carcinoembryonic antigen (CEA) and carbohydrate antigen 153 (CA153) were only 15.08 and 21.1 %, respectively. Remarkably, serum miR-195 had higher sensitivity, 73.97 % (108/146), as a tumor marker in the diagnosis of early stage BC [ductal carcinoma in situ, tumor-node-metastasis (TNM) I, II] compared with the conventional tumor markers CA153 and CEA (12.41 and 7.59 %). Moreover, compared with CEA and CA153, miR-195 had a higher sensitivity for detecting the response to neoadjuvant chemotherapy and significantly increased, more than twofold, after neoadjuvant chemotherapy (P = 0.025, paired t test) in 52.381 % (11/21) of BC cases. However, there were no significant relationships between miR-195 expression and other clinicopathological parameters (TNM stage/pathotype/ER/PR/lymph node status). Our data indicate serum miR-195 is a promising tumor marker for BC diagnosis and general screening, especially for early stage BC. The high sensitivity of miR-195 to neoadjuvant chemotherapy may lay the foundation for future studies on the use of miRNA-based methods for monitoring BC treatment and therapy.

Comparison of conventional RT-PCR, reverse-transcription loop-mediated isothermal amplification, and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches

Bovine viral diarrhea virus (BVDV) can contaminate biological products produced in bovine or porcine cells or manufactured using bovine sera. A rapid, specific, sensitive, and practical method of detecting BVDV in bio-products is needed. The purpose of this study was to compare three assays with respect to their ability to accurately detect BVDV in biological samples, namely reverse-transcription loop-mediated isothermal amplification (RT-LAMP), SYBR green I-based real-time RT-PCR, and conventional RT-PCR. All assays detected BVDV nucleotide and differentiated between BVDV-free and -contaminated bovine sera successfully. In addition, the results were specific to BVDV: the amplification of samples containing the closely related classical swine fever virus or other pathogenic bovine viruses yielded negative results. The lowest detection threshold, 101 copies, was displayed by the SYBR green I-based real-time RT-PCR and RT-LAMP assay. This assay was also the most effective in the detection of BVDV contamination in a set of commercially available bovine sera. The field conditions suggest that RT-LAMP is specific and sensitive to detecting BVDV in biological samples and may be used for quality control of biomaterials.

Epigenetic Modifications in the Development of Atherosclerosis

Introduction: Epigenetics is playing a decisive role in the regulation of gene expression. Furthermore, pathological epigenetic changes have already been associated with various diseases, especially cancer. However, few data are so far available about epigenetic modifications in atherosclerotic lesions. Therefore, we investigated histone methylation and acetylation in different stages of atherosclerosis in patients with carotid artery stenosis. Methods: Carotid plaques (n 1⁄4 120) from our biobank were classified histologically according to AHA and divided as early (type IeIII, n 1⁄4 60) or advanced (type VeVII, n 1⁄4 60) stage of atherosclerosis. Twelve healthy vessels served as controls. Expression of histone methyl and acetyltransferases were analysed by SYBRgreen-based real-time RT-PCR. Immunohistochemistry and western blotting were performed to quantify expression at protein level and to associate histone methylation with the individual cells within the atherosclerotic plaques. Results: In atherosclerosis, methylation of H3K4 was unaltered. In contrast methylation of H3K9 and H3K27 were significantly decreased, compared to control arteries. Expression of H3K4 was increased and H3K9 decreased in smooth muscles, whereas H3K9 and H3K27 were reduced in inflammatory cells in advanced versus early atherosclerosis. MML2 and G9a significantly increased in advanced versus early atherosclerosis, and a significant decrease in expression of G9a was observed between controls and early stages. Increased histone acetylation was observed on H3K9 and H3K27 in SMCs in advanced atherosclerotic lesions compared to healthy vessels, acetylation of H3 at position K9 in SMCs and macrophages was associated with plaque progression. Expression of acetyltransferase GCN5L and MYST1 correlated with severity of atherosclerosis. Conclusion: The extent of histone methylation and acetylation, as well the expression of corresponding transferases were significantly altered in atherosclerotic lesions and were significantly associated with the progression of atherosclerosis, thus suggesting an important contribution of epigenetic mechanisms to plaque progression and vulnerability.

A SYBR Green-based Real Time RT-PCR Assay for Detection of the Emerging H7N9 Virus

A SYBR Green-based Real Time RT-PCR Assay for Detection of the Emerging H7N9 Virus