Analysis of gene expression

The field of gene expression analysis has only recently become of broader interest to users of mass spectrometry. The reason for this lag in interest was simply the lack of methods allowing the absolute quantification of NAs in general, or mRNA in particular. Mass spectrometry only allows an endpoint analysis, and even then a standard for normahzation (such as a second allele) is required. Hence, the approaches used have focused on the semi-quantitative analysis of allelic expression which, as described above, is a rather narrow-albeit expanding-field of research. 

In a different approach. Ding and Cantor recently described the use of an internal standard added to cDNA samples to enable the quantification of NAs with technologies lacking real-time monitoring capabilities (as are employed for realtime PCR) [225]. This approach uses a synthetic oligonucleotide designed to match 

The post-PCR primer extension reaction targets the nucleotide difference between the internal standard and the cDNA, and generates two specific products that resemble the same reaction performed for aUele-ffequency determination. Analysis of the peak areas allows not only for a relative comparison of cDNA amount versus internal standard, but also for an absolute quantification, as the concentration of the internal standard is known. The use of an internal standard with the same sequence as the target sequence alleviates common issues related to quantification. Due to the same PCR amplification efficiency, the process is PCR-cycle-independent, and real-time monitoring of the amplification process becomes obsolete, as it will impact on both the internal standard and the cDNA in the same way. 

The description by Ding and Cantor is very recent, and further use of the proposed method in various laboratories will demonstrate the utility and accuracy of the approach. The competitor PCR approach described by Ding and Cantor against real-time PCR (rtPCR) was validated in a more recent report from Elvidge et al., who demonstrated a good correlation between real-time PCR data and MALDI-TOF-MS data [226]. There was, however, a significant difference in sensitivity, with low-abundant mRNAs being much more readily detected and quantified by the 

The basic concept described by Ding et al. has recently been extended to exciting new aspects of gene expression, including the quantitation of allele-specific expression and the quantitative analysis of splice variants [227, 228). 

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DNA microarrays, or DNA chips consist of thousands of individual DNA sequences arrayed at a high density on a single matrix, usually glass slides or quartz wafers, but sometimes on nylon substrates. Probes with known identity are used to determine complementary binding, thus allowing the analysis of gene expression, DNA sequence variation or protein levels in a highly parallel format. 

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Apart from SR-BI, SR-BII, CD36, and ABCA1, a microarray analysis of gene expression in human RPE reveals some additional lipid transporters that might potentially be involved in intracellular transport of carotenoids and/or their efflux from the RPE cells into the neural retina or out of the retina into the choroidal blood (van Soest et al., 2007). These include other ABC... 

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We thank Dr. Jacky Snoep for collaborating in the time scale analysis of gene expression and metabolism, Douglas Kell for helpful discussions, and the Commonwealth of Virginia for financial support. 

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Analysis of Gene Expression in the Brain Using Differential Display... 

Analysis of gene expression in the brain using differential display... 

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