Absolute quantification of gene expression in biomaterials research using real-time PCR

Biomaterials (Impact Factor: 8.56). 01/2007; 28:203-210.


Cited By (since 1996): 18, Export Date: 27 September 2011, Source: Scopus

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Available from: Fook Tim Chew, Jul 17, 2015
    • "This method requires similar amplification efficiencies (see below) for all samples and standards. Therefore, the standard curve template must be carefully chosen (Dhanasekaran et al., 2010; Leong et al., 2007; Malorny et al., 2008; Whelan et al., 2003). Relative quantification is used to estimate changes in gene expression. "
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    ABSTRACT: Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported.
    Food Microbiology 08/2011; 28(5):848-61. DOI:10.1016/j.fm.2011.02.008 · 3.33 Impact Factor
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    • "For HBV-associated HCC cases, HBV-DNA levels in the tumor and its corresponding peritumoral noncancerous tissues were determined by fluorescence quantitative real-time PCR. Absolute quantification analysis was applied and the standard curves were generated as previously described (Leong et al., 2007). The primer sequences of HBV S gene (HBs) and GAPDH are given in Table 2C. "
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    ABSTRACT: A remarkable feature of HBV-associated HCC is male predominance. The cooperation of hepatitis B virus X protein (HBx) with androgen receptor (AR) signaling pathway has been documented to contribute to this dominance. HBx, a multifunctional viral regulator, has been documented to induce promoter hypermethylation and low expression of tumor suppressor genes via activation of DNA methyl-transferase (DNMT) in hepatocarcinogenesis. In prostate cancer, hypermethylation of AR promoter is associated with loss of AR expression. However, the relationship among HBx, DNMTs, the methylation status of AR and AR expression in HBV-associated HCC is still unknown. In this report, we found that HBx correlated with high levels of AR in HCC cases and induced AR expression by stimulating its transcription in liver cell lines. HBx correlated with high expression of DNMTs in HCC cases too. Both in vivo and in vitro, however, the expression of AR was not associated with its promoter methylation status, and the methylation status of AR was not regulated by DNMTs. AR expression is higher in peritumoral tissues than in tumors, as well as being higher in HBV-associated HCC than in HBV-negative cases. Therefore, HBx-induced high expression of AR plays a role during hepatocarcinogenesis, but is not involved with its promoter methylation or DNMTs. HBx-mediated DNMT deregulation is gene-specific, and the expression and methylated regulation of AR is tissue-specific.
    Histology and histopathology 01/2011; 26(1):23-35. · 2.10 Impact Factor
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    • "The quantitative real-time PCR used in this study followed the protocol developed by Leong et al. (2007), for total mRNA quantification . In brief, real-time PCR was performed using 1 mg of total Table 1 Cell surface marker expression in human bone marrow-derived MSCs and human adipose tissue-derived MSCs. "
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    ABSTRACT: High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.
    Journal of Molecular Cell Biology 08/2010; 2(4):199-208. DOI:10.1093/jmcb/mjq011 · 6.77 Impact Factor
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