China
Africa
Explore chapters and articles related to this topic
Toxicogenomic and Toxicoproteomic Approaches for Biomarkers
Published in Anthony P. DeCaprio, Toxicologic Biomarkers, 2006
The powerful PCR technique rapidly became incorporated into other genomic approaches in addition to its direct use for analyzing expression of individual genes. One of these applications was in a method called differential display of mRNA expression (33). This approach to genomic screening exploited a variation of multiplex PCR and was used both to globally examine patterns of altered gene expression and for direct gene discovery. The technique is based on use of short random hexamer primers that simultaneously amplify numerous segments of complementary DNA (cDNA) in the entire isolated RNA pool. Parallel amplification of RNAs from control and treated samples is followed by running out the PCR products on a sequencing gel for a side-by-side pattern comparison of the two samples. Typically, it was observed that most bands were identical in the two samples, indicating that most mRNAs were not differentially expressed (Fig. 4). This observation also provides a means for normalizing the treated to the control pattern, such that the altered band intensities are more apparent, and represent only those products that are substantially up or downregulated by a given treatment. The mRNA segment of interest can then be identified by subsequent isolation of the band from the gel, followed by reamplification, subcloning, and sequencing of the product. Figure 4 shows an example of the results of a differential display experiment from our laboratory, examining the effects of cadmium on gene expression in the zooplankton, Daphnia pulex. Using this method, we identified several candidate mRNA fragments that were up- or downregulated in response to nonovertly toxic exposures of Daphnia to cadmium. As expected, one of these fragments turned out to represent the mRNA for Daphnia metallothionein, but several other cadmium-responsive mRNAs were also identified.
The transcriptomic revolution and radiation biology
Published in International Journal of Radiation Biology, 2022
In the early 1990s the introduction of RT-PCR-based differential display techniques provided another technical boost to studies of differentially expressed genes (Liang and Pardee 1992). More radiation responsive genes were reported, but because cloning and sequencing was required to determine the identity of individual hits, progress remained slow, with most studies describing only one or a few new radiation-induced genes and following a reductionist approach to their study (Gomez et al. 1996; Yan et al. 1996; Chang-Liu and Woloschak 1997; Goltry et al. 1998; Noel et al. 1998; Okamura et al. 2001). While the earlier focus had been on genes with increased expression following DNA damage, differential display experiments also started to identify genes with decreased abundance after irradiation (Woloschak et al. 1995; Paunesku et al. 2000; Watson et al. 2000; Zhou and Rigaud 2001). As an increasingly complex picture of the transcriptional response to DNA damage began to emerge, it became clear that multiple cellular processes, including apoptosis (Paunesku et al. 2000; Okamura et al. 2001), cell cycle regulation (Gomez et al. 1996; Zhou and Rigaud 2001), and cellular signal transduction pathways (Yan et al. 1996; Watson et al. 2000) could be impacted at the level of mRNA abundance.
The role of prostate cancer antigen 3 (PCA3) in prostate cancer detection
Published in Expert Review of Anticancer Therapy, 2018
Guillaume Ploussard, Alexandre de la Taille
In 1999, the differential display clone 3 (renamed PCA3 to reflect its association with PCa) was identified by comparing mRNA expression patterns between tumor and adjacent non-neoplastic tissues [1]. PCA3 gene is located on the long arm of chromosome 9 and is not translated into protein due to various codons. Its function remains unknown. In the original report, Bussmakers et al. demonstrated that the PCA3 gene was highly expressed in prostate tumor cells, and subsequent studies confirmed this differential expression in malignant tissue [2,3]. The PCA3 overexpression has been evaluated at 34-fold higher in malignant cells versus 6-fold in non-malignant tissue [2]. More recent studies have demonstrated that in human beings, there was no significant difference in PCA3 scores in men with chronic prostatitis and high grade prostatic intraepithelial neoplasia compared with other men with negative biopsies [4]. It also has been demonstrated that the variability of PCA3 scores on repeated measures at an individual basis confirmed the risk class for about 80% of patients with an upgrading in two thirds and a downgrading in one third of remaining cases. Thus, given its promising value in PCa field, this gene and its non-coding mRNA have been further studied as potential biomarker for PCa detection and prognosis assessment. A RT-PCR-based PCA3 test was developed and became commercially available to evaluate the utility of PCA3 to detect PCa cells in urine samples [5]. Prostatic cells are released into urine after a prostatic massage. Approximately, 20–30 ml of first-catch urine is required for the test after at least three strokes to each prostate lobe between a DRE. Then, the expression of PCA3 mRNA is integrated into a ratio taking into account a housekeeping gene with a relatively constant expression in prostate cells. The PSA gene expression has been chosen, and the PCA3 score is calculated by the ratio between PCA3 mRNA copies/ml to PSA mRNA copies/ml, multiplied by 1000. The result provided by this formula is a continuous variable. For clinical purposes, a clinically validated cut-off has been used in different studies, varying between 20, 25, or 35. A PCA3 cutoff score of 35 is generally used. However, most recent studies show that a lower cut-off score of 25 might be preferable, particularly if a better negative predictive value is needed [5,6]. The technology became simple, reproducible and sensitive when that initial RT-PCR was translated to a transcription-mediated amplification [7]. This test has been proposed to help clinicians decide whether or not performing prostate biopsies.