Article

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

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

ABSTRACT

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

Download full-text

Full-text

Available from: Fook Tim Chew, Jul 17, 2015
  • Source
    • "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: 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.
    Full-text · Article · Feb 2015 · Molecular and Cellular Endocrinology
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    No preview · Article · Aug 2011 · Food Microbiology
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jan 2011 · Histology and histopathology
Show more