Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 4

real-time PCR Related Publications

4 Comparison of Real-Time PCR and FTIR with Chemometrics Technique in Analysing Halal Supplement Capsules

Authors: Mohd Sukri Hassan, Ahlam Inayatullah Badrul Munir, M. Husaini A. Rahman

Abstract:

Halal authentication and verification in supplement capsules are highly required as the gelatine available in the market can be from halal or non-halal sources. It is an obligation for Muslim to consume and use the halal consumer goods. At present, real-time polymerase chain reaction (RT-PCR) is the most common technique being used for the detection of porcine and bovine DNA in gelatine due to high sensitivity of the technique and higher stability of DNA compared to protein. In this study, twenty samples of supplements capsules from different products with different Halal logos were analyzed for porcine and bovine DNA using RT-PCR. Standard bovine and porcine gelatine from eurofins at a range of concentration from 10-1 to 10-5 ng/µl were used to determine the linearity range, limit of detection and specificity on RT-PCR (SYBR Green method). RT-PCR detected porcine (two samples), bovine (four samples) and mixture of porcine and bovine (six samples). The samples were also tested using FT-IR technique where normalized peak of IR spectra were pre-processed using Savitsky Golay method before Principal Components Analysis (PCA) was performed on the database. Scores plot of PCA shows three clusters of samples; bovine, porcine and mixture (bovine and porcine). The RT-PCR and FT-IR with chemometrics technique were found to give same results for porcine gelatine samples which can be used for Halal authentication.

Keywords: Gelatin, real-time PCR, halal, FTIR and chemometrics

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3 Analysis of OPG Gene Polymorphism T245G (rs3134069) in Slovak Postmenopausal Women

Authors: J. Bernasovská, J. Poráčová, I. Boroňová, E. Petrejčiková, J. Kľoc, Z. Tomková, S. Mačeková, M. M. Blaščáková

Abstract:

Osteoporosis is a common multifactorial disease with a strong genetic component characterized by reduced bone mass and increased risk of fractures. Genetic factors play an important role in the pathogenesis of osteoporosis. The aim of our study was to identify the genotype and allele distribution of T245G polymorphism in OPG gene in Slovak postmenopausal women. A total of 200 unrelated Slovak postmenopausal women with diagnosed osteoporosis and 200 normal controls were genotyped for T245G (rs3134069) polymorphism of OPG gene. Genotyping was performed using the Custom Taqman®SNP Genotyping assays. Genotypes and alleles frequencies showed no significant differences (p=0.5551; p=0.6022). The results of the present study confirm the importance of T245G polymorphism in OPG gene in the pathogenesis of osteoporosis.

Keywords: Osteoporosis, real-time PCR, OPG gene, T245G polymorphism

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2 Gene Expressions Associated with Ultrastructural Changes in Vascular Endothelium of Atherosclerotic Lesion

Authors: M. Maimunah, G.A. Froemming, H. Nawawi, M.I. Nafeeza, O. Effat, M.R. Rohayu Izanwati, M.S. Mohamed Saifulaman

Abstract:

Attachment of the circulating monocytes to the endothelium is the earliest detectable events during formation of atherosclerosis. The adhesion molecules, chemokines and matrix proteases genes were identified to be expressed in atherogenesis. Expressions of these genes may influence structural integrity of the luminal endothelium. The aim of this study is to relate changes in the ultrastructural morphology of the aortic luminal surface and gene expressions of the endothelial surface, chemokine and MMP-12 in normal and hypercholesterolemic rabbits. Luminal endothelial surface from rabbit aortic tissue was examined by scanning electron microscopy (SEM) using low vacuum mode to ascertain ultrastructural changes in development of atherosclerotic lesion. Gene expression of adhesion molecules, MCP-1 and MMP-12 were studied by Real-time PCR. Ultrastructural observations of the aortic luminal surface exhibited changes from normal regular smooth intact endothelium to irregular luminal surface including marked globular appearance and ruptures of the membrane layer. Real-time PCR demonstrated differentially expressed of studied genes in atherosclerotic tissues. The appearance of ultrastructural changes in aortic tissue of hypercholesterolemic rabbits is suggested to have relation with underlying changes of endothelial surface molecules, chemokine and MMP-12 gene expressions.

Keywords: real-time PCR, Ultrastructure of luminal endothelial surface, Macrophage metalloelastase (MMP-12)

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1 A Novel Multiplex Real-Time PCR Assay Using TaqMan MGB Probes for Rapid Detection of Trisomy 21

Authors: Mehrdad Hashemi, Mitra Behrooz Aghdam, Reza Mahdian, Ahmad Reza Kamyab

Abstract:

Cytogenetic analysis still remains the gold standard method for prenatal diagnosis of trisomy 21 (Down syndrome, DS). Nevertheless, the conventional cytogenetic analysis needs live cultured cells and is too time-consuming for clinical application. In contrast, molecular methods such as FISH, QF-PCR, MLPA and quantitative Real-time PCR are rapid assays with results available in 24h. In the present study, we have successfully used a novel MGB TaqMan probe-based real time PCR assay for rapid diagnosis of trisomy 21 status in Down syndrome samples. We have also compared the results of this molecular method with corresponding results obtained by the cytogenetic analysis. Blood samples obtained from DS patients (n=25) and normal controls (n=20) were tested by quantitative Real-time PCR in parallel to standard G-banding analysis. Genomic DNA was extracted from peripheral blood lymphocytes. A high precision TaqMan probe quantitative Real-time PCR assay was developed to determine the gene dosage of DSCAM (target gene on 21q22.2) relative to PMP22 (reference gene on 17p11.2). The DSCAM/PMP22 ratio was calculated according to the formula; ratio=2 -ΔΔCT. The quantitative Real-time PCR was able to distinguish between trisomy 21 samples and normal controls with the gene ratios of 1.49±0.13 and 1.03±0.04 respectively (p value <0.001). These results represent the presence of 3 copies of target gene in DS samples Vs 2 copies in normal controls. The results of quantitative Real-time PCR were in complete agreement with results of cytogenetic analysis. This study confirms previous reports regarding successful implementation of quantitative Real-time PCR for detection of trisomy 21. However, the assay has been improved by using MGB probes and more accurate data analysis. This assay, in particular, when performed in combination with another molecular assay such as QF-PCR or MLPA, can be used as a reliable technique for rapid prenatal diagnosis of trisomy 21.

Keywords: Trisomy 21, real-time PCR, MGB-TaqMan Probes, Gene Dosage

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