Transcript Survey Techniques

Early attempts to build gene expression databases relied on Northern blotting, RNase protection, RT-PCR, in situ hybridization, and nuclease protection strategies. For the most part, these methodologies are limited by low throughput and their laborintensive nature. More recent strategies have included the use of subtractive hybridization and differential display. However, these protocols are labor intensive and cost prohibitive. They also suffer from varied false positive rates and a general lack of sensitivity, rendering it difficult to identify a subset of genes and gene products involved in processes key to normal or aberrant development. To date, these technical factors have precluded a genome-wide scan to elucidate the sperm transcriptome. However, techniques that afford genome-wide scans, such as Serial Analysis of Gene Expression (SAGE), permit one to identify genomic heterogeneity that underlies the developmental pathways specific to individual cells, tissues, and organ systems.29 While in some cases SAGE may provide a sensitive means of detecting RNA species, the sequences defined by SAGE can be unknown "snapshots" of a complete sequence. Even though this is a powerful technology, this sequencing-intensive process is comparatively slow and relatively expensive, and thus has not become widely employed.

The potential of microarrays to address key issues of development and differentiation was immediately realized.30-32 In a single experiment, this technology permits the simultaneous determination of the expression of thousands of genes. This enables the construction of detailed expression and genetic profiles.33-35 Recent microarray-based ovarian and breast cancer studies have demonstrated both the potential diagnostic and prognostic value of this method.36,37 It is also clear that this technology

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Figure 6.3 Experimental design. Human ejaculates were obtained from 10 healthy volunteers of proven fertility. Nine of the samples were pooled then purified using two sequential centrifugations through 40:80 discontinuous Percoll gradients. The purity and integrity of both preparations of spermatozoal RNAs were electro-phoretically assessed and verified by RT-PCR using intron spanning protamine-2 primers. RNA from pooled histologically normal human testes was purchased from Clontech Laboratories, Inc. (Palo Alto, CA, USA). Probes were prepared from the testes and spermatozoal RNAs by reverse-transcription from the total, poly(A+) sperm RNAs and total testes RNA, then individually hybridized to the arrays.

is well suited to the toxicological arena15 and a new field of toxicogenomics has been created.38 One of the early applications of this technology was the classification of toxicants based on the responsive profile of the transcriptome.39 This has since been reviewed.40

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