A flexible biosensor has been developed that utilizes immobilized nucleic acid aptamers to specifically detect free nonlabeled non-nucleic acid targets such as proteins. In a model system, an anti-thrombin DNA aptamer was fluorescently labeled and covalently attached to a glass support. Thrombin in solution was selectively detected by following changes in the evanescent-wave-induced fluorescence anisotropy of the immobilized aptamer. The new biosensor can detect as little as 0.7 amol of thrombin in a 140-pL interrogated volume, has a dynamic range of 3 orders of magnitude, has an inter-sensing-element measurement precision of better than 4% RSD over the range 0-200 nM, and requires less than 10 min for sample analysis. The aptamer-sensor format is generalizable and should allow sensitive, selective, and fast determination of a wide range of analytes.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in both plants and animals. miRNA genes have been implicated in a variety of important biological processes, including development, differentiation, apoptosis, fat metabolism, viral infection, and cancer. Similar to protein-coding messenger RNAs, miRNA expression varies between tissues and developmental states. To acquire a better understanding of global miRNA expression in tissues and cells, we have developed isolation, labeling, and array procedures to measure the relative abundance of all of the known human mature miRNAs. The method relies on rapid isolation of RNA species smaller than 40 nucleotides (nt), direct and homogenous enzymatic labeling of the mature miRNAs with amine modified ribonucleotides, and hybridization to antisense DNA oligonucleotide probes. A thorough performance study showed that this miRNA microarray system can detect subfemtomole amounts of individual miRNAs from <1 mg of total RNA, with 98% correlation between independent replicates. The system has been applied to compare the global miRNA expression profiles in 26 different normal human tissues. This comprehensive analysis identified miRNAs that are preferentially expressed in one or a few related tissues and revealed that human adult tissues have unique miRNA profiles. This implicates miRNAs as important components of tissue development and differentiation. Taken together, these results emphasize the immense potential of microarrays for sensitive and high-throughput analysis of miRNA expression in normal and disease states.

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In nematodes, the RNA products of some genes are trans-spliced to a 22-nucleotide spliced leader (SL), while the RNA products of other genes are not. In Caenorhabditis ekgans, there are two SLs, SL1 and SL2, donated by two distinct small nuclear ribonucleoprotein particles in a process functionally quite similar to nuclear intron removal. We demonstrate here that it is possible to convert a non-trans-spliced gene into a trans-spliced gene by placement of an intron missing only the 5' splice site into the 5' untranslated region. Stable transgenic strains were isolated expressing a gene in which 69 nucleotides of a vit-5 intron, including the 3' splice site, were inserted into the 5' untranslated region of a vit-21vit-6 fusion gene. The RNA product of this gene was examined by primer extension and PCR amplification. Although the vit-21vit-6 transgene product is not normally trans-spliced, the majority of transcripts from this altered gene were trans-spliced to SL1. We termed the region of a trans-spliced mRNA precursor between the 5' end and the first 3' splice site an "outron." Our results suggest that if a transcript begins with intronlike sequence followed by a 3' splice site, this alone may constitute an outron and be sufficient to demarcate a transcript as a trans-splice acceptor. These findings leave open the possibility that specific sequences are required to increase the efficiency of trans-splicing.In both trypanosomes and nematodes, mRNAs are present which begin with an untranslated leader sequence acquired by trans-splicing between a spliced leader (SL) small nuclear ribonucleoprotein particle (snRNP) and recipient transcripts (7,17,20,24,32,35, 36). Trypanosome pre-mRNAs do not contain introns, and all begin with the SL (21). In contrast, both trans-splicing and intron removal are occurring on the same nematode transcripts, and only a subset of Caenorhabditis elegans genes (estimated at about 15% [1]) specify transcripts that receive an SL (4). Hence, the interplay between conventional cis-splicing and transsplicing in nematodes is an intriguing and largely unexplored area. It is clear that the two processes are quite closely related: consensus sequences for the 3' splice site of introns and trans-splice sites are the same and both occur via a 2'-5' branch (or lariat) intermediate (2,35). Furthermore, the donor in the trans-splicing reaction, a 100-nucleotide RNA called SL RNA, occurs in the form of an snRNP. It has the trimethylguanosine cap typical of those snRNPs involved in the catalysis of cis-splicing and is bound by some of the same immunologically defined proteins (7,35, 36 pairs of very similar genes have been described in which only one is trans-spliced (9,19,20,26). In some of these cases, the coding regions of the gene pairs are nearly identical. Thus, it is likely that the information for transsplicing is in the region upstream of the trans-splice site. However, a comparison of DNA sequences upstream from the trans-splice acceptor sites of 11 genes which receive SLi (9,13,14,16,19,20,26) a...

Context.—Expression profiling by microarrays and real-time polymerase chain reaction–based assays is a powerful tool for classification and prognostication of disease; however, it remains a research tool, largely reliant on frozen tissue. Limiting the utility of expression profiling is the isolation of quality nucleic acids from formalin-fixed, paraffin-embedded tissue. The collection, handling, and processing of tissue directly impacts the biomolecules that can be recovered from it. High-quality nucleic acids can be obtained from formalin-fixed, paraffin-embedded tissue, but greater attention to all steps in the process of tissue handling and preparation is required. Objective.—To summarize the current state-of-the-art of preanalytic factors in tissue handling and processing as they impact the quality of RNA obtainable from formalin-fixed, paraffin-embedded tissue. The goals are to provide recommendations that will improve RNA quality for expression profiling from formalin-fixed, paraffin-embedded tissue and highlight areas for additional research. Tissue is an analyte and it must be handled in a standardized fashion to provide consistent results. Data Sources.—The literature was reviewed. Consultation with industry and academic leaders in the use of RNA for expression profiling was obtained to identify areas for additional research. Conclusions.—Development of RNA-based assays from formalin-fixed, paraffin-embedded tissue is feasible. Greater attention to tissue handing and processing is essential to improve the quality of biospecimens for the development of robust RNA-based assays. Standardization of procedures and vigorous testing of alternative protocols are required to ensure that these assays function as designed.