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ANALYTICAL CURRENTS Microarrays for monitoring alternative splicing The researchers modeled data for each transcript, assuming all exons were present. The difference between the expected and the experimentally observed exon junctions was used to predict whether the mRNA was alternatively spliced in the tissue or cell line tested. Predictions based on the microarray data were validated by reverse–transcriptase PCR and sequencing. For example, in tissues in which alternative splicing was predicted for the gene OCRL1, two known alternative transcripts were found. Johnson, Shoemaker, and colleagues also discovered new cases of alternative splicing by using microarrays, such as a novel variant of the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase transcript. The microarray method is more sensitive than analyzing expressed sequence tag (EST) data because EST libraries are biased toward highly abundant mRNAs and transcript termini. Although previous data from EST analyses revealed alternative splicing preferences for both transcript ends, the researchers Uterus 50 found the highThyroid Testes 45 est frequency of Spinal cord splicing events at 40 the 5´ end. The Melanoma (G361) 35 process occurred Lung 30 at similar rates Liver throughout the 25 Kidney rest of the tran20 Heart script length. 15 Descending colon According to the researchers, 10 although the miBrain 5 croarray approach Bone marrow is powerful, it 2 4 6 8 10 12 14 16 18 can be improved Junction probes with the addition 3' Full length 5' of probes for se1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Variant 5' 3' quences within exons and probes Predictions of alternative splicing events at junctions 12 and 13 in the 3-hy- that account for droxy-3-methyl-glutaryl coenzyme A reductase mRNA are shown for differ- all possible exonent tissues. Dark blue boxes indicate a high probability of alternative splic- skipping events. (Science 2003, ing. (Adapted with permission. Copyright 2003 American Association for 302, 2141–2144) the Advancement of Science.) Samples
Splicing rids messenger RNA (mRNA) of introns to produce a transcript containing only exons, which are the coding regions of genes. But sometimes, one or two exons are also spliced out of transcripts, resulting in alternatively spliced forms with different functions. Conventional approaches for monitoring alternative splicing are labor-intensive and expensive, which makes it difficult to characterize transcripts across the entire genome. To better understand the role of alternative splicing across different diseases, tissues, and stages of development, Jason Johnson, Daniel Shoemaker, and colleagues at Merck and Co. developed five microarrays with probes that span ~125,000 different exon–exon junctions. The researchers used the microarrays to monitor alternative splicing in >10,000 human genes in 52 tissues and cell lines. Unlike other methods of analyzing splice junctions, the microarray assay is tissuespecific and is not biased for the detection of splicing at transcript ends.
Direct detection of microRNAs Most nucleic acid detection schemes are indirect, relying on amplification by such methods as PCR to generate a pool of starting material. Increasingly, however, researchers want to analyze the nucleic acids in a tissue or organism directly, without introducing extra steps and possible errors. Michael Famulok and colleagues at Universität Bonn (Germany) are among those developing direct detection assays. They describe a new method for the direct detection of microRNAs (miRNAs), which are small ribonucleic acids that regulate gene expression. The assay has a detection limit of 50 fmol and is at least 10-fold more sensitive than a molecular beacon. Famulok and colleagues modified a ribozyme—an RNA molecule with enzymatic activity—to include a binding domain specific for a certain miRNA. If the cognate miRNA is present, it binds to the new domain and frees the rest of the ribozyme to cleave the substrate. The substrate for the ribozyme has a fluorophore at one end and a quencher at the other end. Fluorescence is detected only when the ribozyme is active. The researchers designed nine ribozymes that could be activated by different miRNAs. For each ribozyme, fluorescence increased upon addition of the cognate miRNA. Only a baseline level of fluorescence was observed when noncognate miRNAs were added. Although the assay is sensitive, Famulok and colleagues report that the stability of the modified ribozymes and the substrates must be enhanced in order to detect nucleic acids in vivo. (J. Am. Chem. Soc. 2004, 126, 722–723)
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