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Wednesday, June 18

Applying Technologies for the “Other” Nucleic Acids RNA, RNAi, mRna, miRNA, and siRNA*

12:30 pm Registration

1:30  Welcome by Workshop Chairperson

1:45   RNAi Activity from siRNA Duplexes Containing Nicks or Gaps in the Passenger Strand
James McSwiggen, Ph.D., Director, Biochemistry & Bioinformatics, Nastech Pharmaceutical Company, Inc.
It has been generally accepted that a continuous siRNA duplex is required for RISC assembly, but we report that siRNAs containing a nick or gap in the passenger strand can also be extremely active in RNAi. We have called these constructs “meroduplexes”, or mdRNA, to highlight the incomplete nature of the siRNA duplex. Meroduplex RNAi activity is maintained with a nick at almost any position along the siRNA passenger strand—as long as the oligos retain sufficient base-pairing free energy to form the duplex. In addition, gaps in the passenger strand of up to four nucleotides still support RNAi, and both RISC and dicer substrate forms are active. This discovery should help eliminate the off-target potential of passenger loading into RISC, and should also help to elucidate the mechanism of RISC loading and passenger strand removal.

2:15  Challenges and Solutions for Detection of Unprecedented Targets
Alexander Aristarkhov, Ph.D., Director, Science Technology, S&M, Exiqon
miRNA is one of the small regulatory RNAs and the key component of overlooked global regulatory networks in basic processes like development and species specific regulation of particular functions belonging to evolutionary distinct organisms. It is an unprecedented target for detection because of small size and similarity and as an active molecule it requires different mRNA approaches. Accuracy, precision, and multiple validations are needed to discover and identify functions of miRNAs. Here we will demonstrate the complete set of LNA-based enabling tools to ensure efficiency of this process.

2:45   Cap-Analysis Gene Expression (CAGE) Analysis of Transcriptional Complexity and Regulation
Piero Carninci, Ph.D.,  Team Leader, Omics Science Center, RIKEN
The genome sequence is an invaluable resource, yet it is still hard to identify all its encoded RNAs and regulatory regions. Despite the fact that there are about 20,000 protein coding genes, 93% of the mammalian genomes are expressed and there are more than 180,000 different mRNAs or non-coding RNAs, which constitute more than 44,000 transcriptional units (TUs). The development of a novel generation of sequencing instruments allows addressing the RNA complexity at a much higher definition. We are using the 454 Life Science, Illumina/Solexa and ABI-SOLiD sequencers to produce hundreds of millions of sequences tags, including the CAGE (cap-analysis gene expression) and short RNA libraries. Sequencing-based transcriptome analysis produces much more detailed and sensitive data than microarrays at a comparable cost. CAGE analysis identifies and maps more than 230,000 core promoters, resulting in a fine redefinition of the promoter structure.  CAGE analysis allows correlating promoter elements to transcription as demonstrated by reconstructing the transcriptional network of cell differentiation.
In the next few years, deep sequencing of functional libraries derived from various RNA classes will allow a broad, quantitative and systematic approach to biological problems.

3:15  Networking Refreshment Breaks

3:45   A Robust Quality Control Standard for Quantitative Detection of mRNA
Z. Lewis Liu, Ph.D., Research Molecular Biologist, National Center for Agricultural Utilization Research, USDA
We previously reported universal RNA controls for gene expression analysis that can be applied to different platforms of microarray and real time qRT-PCR.  Based on this technology, we currently developed a robust master equation derived from the robust quality control standard for detection of mRNA by qRT-PCR.  Using Saccharomyces cerevisiae as an example, we demonstrate its application and consistent performance independent from stress challenged conditions.  This development provided reliable reference for mRNA detection and absolute quantification, simplified the conventional qRT-PCR procedures, and increased data reliability, reproducibility, and throughput of the qRT-PCR assay. 

4:15   Analysis of miRNA Expression Levels from Paraffin-Embedded Formalin-Fixed Tissue Samples
Thorsten Singer, Ph.D., Scientific Associate Director, R&D, Qiagen GmbH
In recent years, miRNAs have received increasing attention as important regulators of protein expression, with important functions e.g. in development, cancer, and other diseases. There are large collections of cancer-related paraffin-embedded formalin-fixed (FFPE) tissue samples, primarily for use histopathological examination. Although RNA for molecular studies can be extracted from such samples, RNA quality is severely impaired. We have developed methods to extract RNA including miRNAs and other small RNA from FFPE samples, and to measure specific miRNA expression levels. We demonstrate that biologically meaningful results can be obtained from FFPE samples. Thereby we show that the accessible information content existing in the large collections of FFPE samples worldwide comprises miRNA as well.

4:45   Close of Pre-Conference Workshop

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