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Article: Absolute quantification of gene expression in biomaterials research using real-time PCR
[Show abstract] [Hide abstract] ABSTRACT: Background: Single nucleotide polymorphisms and variant expression of some interferon (IFN) genes in individuals with chronic hepatitis B virus (HBV) infection might be related to higher viral load and disease complications. Thereby, whole blood samples of 208 patients (94 chronic HBV-infected patients and 114 HBV immune subjects) were analyzed to investigate the association between IFNG (-5A -> G), IFNA1 (-2C -> T) and IFNAR1 (-97T -> C) genes with their expression levels and HBV viral load. Methods: Genotyping was performed by high-resolution melting analysis with quantitative PCR (qPCR). Viral load quantification and gene expression were also carried out using qPCR. Results: Chronic HBV-infected subjects with IFNA1 CT genotype and T allele were more likely to develop protection against HBV when compared to immune subjects with wild-type genotype (IFNA1 CT/CC: OR = 0.45, p = 0.01, and T/C allele: OR = 0.55; p < 0.01). In patients with IFNAR1 wild-type TT genotype, the expression levels of this receptor may explain the lower viral load (r(2) = 0.40; p = 0.04) and protection against chronic infection. Conclusions: These findings suggest that the polymorphic variant of IFNA1 (- 2) gene is associated with chronic HBV infection, and high expression levels of the IFNAR1 gene and low levels of IFNA1 might contribute to the pathogenesis of chronic infection in these subjects. (C) 2016 S. Karger AG, Basel
- "There was no significant difference (p > 0.05) with respect to viral load between ALT, AST and GGT enzymes, cytokine expression levels, and total and indirect bilirubin ( table 2) . The frequencies fit the criteria set out by Manly  in 2005. The genotype distribution in our study did not significantly differ from the distribution expected according to the Hardy-Weinberg equilibrium for the three genes analyzed (p > 0.05). "
[Show abstract] [Hide abstract] ABSTRACT: Herein, we characterized the spatio-temporal expression, cellular distribution and regulation by androgens of the β-defensin SPAG11C, the rat ortholog of the human SPAG11B isoform C, in the developing epididymis by using RT-PCR, in situ hybridization and immunohistochemistry. We observed that Spag11c mRNA was ubiquitously expressed in rat fetuses, but preferentially detected in male reproductive tissues at adulthood. SPAG11C (mRNA and protein) was prenatally mainly detected in the mesenchyme of the Wolffian duct, switching gradually after birth to a predominant localization in the epididymis epithelium during postnatal development. In the adult epididymis, smooth muscle and interstitial cells were also identified as sources of SPAG11C. Furthermore, SPAG11C was differentially immunolocalized on spermatozoa surface during their transit from testis throughout caput and cauda epididymis. Developmental and surgical castration studies suggested that androgens contribute to the epididymal cell type- and region-specific modulation of SPAG11C mRNA levels and immunolocalization. Together our findings provide novel insights into the potential role of β-defensins in the epididymis. Copyright © 2015. Published by Elsevier Ireland Ltd.
- "Absolute quantification of Spag11c mRNA levels was based on Leong et al. (2007). Briefly, cDNA samples (250 ng) from adult caput epididymis were amplified by PCR (40 cycles) in a final volume of 20 μl containing 20 mM Tris–HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 2 U Taq polymerase (Invitrogen) and 0.4 μM of each Spag11c forward and reverse primers (Table 1). "
[Show abstract] [Hide abstract] ABSTRACT: Tissue engineering is essentially a technique for imitating nature. Natural tissues consist of three components: cells, signalling systems (e.g. growth factors) and extracellular matrix (ECM). The ECM forms a scaffold for its cells. Hence, the engineered tissue construct is an artificial scaffold populated with living cells and signalling molecules. A huge effort has been invested in bone tissue engineering, in which a highly porous scaffold plays a critical role in guiding bone and vascular tissue growth and regeneration in three dimensions. In the last two decades, numerous scaffolding techniques have been developed to fabricate highly interconnective, porous scaffolds for bone tissue engineering applications. This review provides an update on the progress of foaming technology of biomaterials, with a special attention being focused on computer-aided manufacturing (Andrade et al. 2002) techniques. This article starts with a brief introduction of tissue engineering (Bone tissue engineering and scaffolds) and scaffolding materials (Biomaterials used in bone tissue engineering). After a brief reviews on conventional scaffolding techniques (Conventional scaffolding techniques), a number of CAM techniques are reviewed in great detail. For each technique, the structure and mechanical integrity of fabricated scaffolds are discussed in detail. Finally, the advantaged and disadvantage of these techniques are compared (Comparison of scaffolding techniques) and summarised (Summary).
- "However, release of acidic degradation products can cause a severe inflammatory response in the body (Bergsma et al. 1993; Tam et al. 1996; Martin et al. 1996; Suuronen et al. 1998; Tatakis and Trombelli 1999; Bostman and Pihlajamaki 2000). Since the 1990s, other types of aliphatic polyester: polyhydroxyalkanoates (PHA) particularly poly-3- hydroxybutyrate (P3HB), copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly-4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxy- hexanoate (PHBHHx) and poly-3-hydroxyoctanoate (Leong et al. 2007 ) have been increasingly investigated as scaffolding materials for tissue engineering application due to their high biocompatibility (Chen and Wu 2005; Misra et al. 2006). They are natural thermoplastic polyesters produced by a wide variety of microorganisms under imbalanced growth conditions (Doi et al. 1995; Li et al. 2005). "