Next Generation siRNA Technology for Improved Silencing Efficacy, Potency, and Specificity and Cleaner RNAi Results

新一代siRNA的技術與應用

Speaker:
Mr. David Dorris, Ph. D.
General Manager,
RNAi and Cell Biology
Applied Biosystems

Historically, RNA interference has enjoyed mixed successes. While knockdown may be good, reducing mRNA expression levels by more than 70%, it suffered from problems with off-target knockdown and unclear phenotypic results. Ambion has changed this with the Silencer® Select Pre-designed and Validated siRNAs. Using a supervised computer learning method based on enormous empirical data sets, Ambion’s algorithm development takes a classification approach rather than a linear regression approach, and results in the ability to differentiate “hyperfunctional” versus mediocre siRNAs. The new sequences are then put through a series of rigorous tests, including a proprietary toxicity classifier and several microRNA filters to reduce potential off-target effect. After the new sequence development and filters, we added a novel chemical modification to improve strand bias and further reduce the small chance that there might be off-target effects. But the work wasn’t complete until the phenotypic effects were examined. In this presentation, we show data demonstrating the reduced impact on the cholesterol biosynthesis, apoptotic and cell cycling pathways. Come see how Silencer Select siRNAs can help you with your gene expression research!

A novel approach for miRNA profiling, discovery and whole transcriptome profiling using massively parallel ligation-based dibase sequencing technology

細胞微核醣核酸全體解碼與表現定量的最新技術平台

Next-generation sequencing (NGS) platforms produce 10-100’s of millions of short reads (25-50bp) in a single run which makes them particularly suited for tag counting application including gene expression profiling. Using a newly developed library protocol which requires low sample input and results in sequence ready samples in less than a day, we explored the expression profiles of small non-coding RNAs in two normal tissues, using the SOLiD™ system. Applied Biosystem’s new NGS platform. The frequency and distribution of miRNAs, isomiRNAs and miRNA* were evaluated and the fold changes generated from these tissues were compared to those of 380 TaqMan® miRNA assays.  Significant correlation levels were observed confirming this approach for small RNAs expression profiling.  Moreover, more than 3000 potentially novel miRNAs or non-coding RNAs were discovered.We also report on transcript coverage, detection and correlation with microarrays from a data set using this approach to analyze gene expression profiles in the samples used by the MAQC project.

This new library approach coupled with the SOLiD™ System provides a high throughput method for digital gene expression that enables the discovery of novel small ncRNAs and miRNAs as well as profiling their expression levels, without the probe bias of microarrays.

即刻 報名參加 研討會, 一同探討 siRNA, microRNA 與 mRNA 的新技術在 RNA 研究的進展！會中另舉辦抽獎活動，凡完整填答問卷，將有機會抽中 iPOD Touch.

Advancing RNA Amplification and Labeling for Gene Expression Profiling

如何突破mRNA microarray技術中RNA增殖與標識的瓶頸

Speaker:
Tom Bittick
Senior Product Manager, Expression Analysis
Applied Biosystems

For more than 7 years, Ambion scientists have had dedicated programs aimed to commercialize and standardize the critical reagents involved in microarray chemistries including sample preparation, amplification, labeling, RNA standards, and hybridization/washing/staining buffers. The latest development has resulted in the release of the next generation MessageAmp Premier RNA Amplification kit for the preparation of biotin-labeled RNA samples for Affymetrix GeneChip® microarray analysis. This kit builds upon the proven gold standard methodology for high density microarray experimentation based on the original T7 in vitro transcription amplification technology, also known as the Eberwine or the RT-IVT method (Van Gelder et al. 1990). Each step has been extensively optimized resulting in an overall improvement in microarray sample processing, including reduced input (as little as 20ng), reduced time to amplification and even a simplified workflow that, due to the magnetic bead-based purification, allows for high throughput and robotic applications.