NCode TM miRNA Analysis Platform Identifies Differentially Expressed Novel miRNAs in Adenocarcinoma Using Clinical Human Samples Provided By BioServe. Mason Brooks 1, Mark Landers 1, Dan Krissinger 1, Vipin Adhlakha 2, Mike Seddon 2 Kristin Wiederholt 1, and Christopher Adams 1 Invitrogen Corporation, Carlsbad CA 1 and BioServe, Beltsville MD 2 AbstractNCode TM miRNA Profiling Workflow Novel miRNA expression in adenocarcinoma Conclusions NCode TM Rapid Labeling qRT-PCR validation of novel miRNA expression MicroRNAs (miRNAs) are nucleotide non- coding RNAs that regulate gene expression by inhibiting translation or triggering degradation of specific mRNA targets. miRNAs appear to play a critical role in directing cellular differentiation and have been implicated in several disease states. The NCode™ Rapid miRNA Labeling System, in conjunction with the NCode ™ miRNA Microarrays, provides a simple and rapid method to profile miRNA expression in any given cell type. The NCode TM Human miRNA Microarray contains the most up-to- date content available in Sanger and hundreds of novel miRNAs discovered through deep sequencing. Here we will describe how global miRNA expression can be profiled in minimal amounts of Trizol- extracted total RNA and their individual expression validated using a novel quantitative, real-time SYBR® Green RT-PCR assay. Using samples from BioServe’s Global Repository of clinical tissue samples and BioServe’s MAPS (isolating sample DNA, RNA and protein), we profiled the miRNA expression in matched, adenocarcinoma and normal colon samples and observed differential expression of novel miRNAs, suggesting possible biological relevance of these novel miRNA sequences in tumorigenesis. The NCode™ miRNA Analysis platform, paired with high quality BioServe samples, is a powerful method for identifying disease related miRNAs, which may serve as candidates for diagnostic or therapeutic targets. Background Experimental Overview NCode TM Human miRNA microarray v3 I.Evolutionarily Conserved II.Estimated ,00 III. Interaction with mRNA target suppressing protein expression  Perfect-mRNA degradation  Imperfect-translational suppression IV. Influence most human protein-coding genes  1/3 mRNAs targeted by miRNAs miRNA biogenesis  Novel miRNA sequences were detected in colon tissues by miRNA array, and validated by qRT-PCR.  Distinct novel miRNA expression patterns were observed between normal and tumor samples.  The NCode TM platform, including the novel miRNA content of the NCode TM miRNA microarray, is a powerful tool for the detection and analysis of miRNAs. Product Information NCode™ Rapid miRNA Labeling System – no miRNA enrichment needed (MIRLSRPD-20) NCode™ Human miRNA Microarray v3- up-to-date array content based on Sanger miRBASE version 10.0 plus novel content (MIRAH3-05) NCode TM miRNA qRT-PCR Universal cDNA synthesis - flexibility (MIRQER-100) NCode TM miRNA Linear Amplification System- High fidelity amplification from small amounts of RNA (MIRAS-20) NEW NCode™ Profiler- free desktop software for experimental planning and data analysis miRNA Expression Profiling  Obtained matched, cancer vs. normal colon, total RNA patient samples from BioServe.  Identified expression of novel miRNAs by NCode TM microarray using NCode TM Profiler software.  Validation by qRT-PCR. Novel miRNAs found to be differentially expressed in adenocarcinoma by microarray, were validated by NCode TM qRT-PCR. The figures above compare fold change values obtained by microarray to values by qRT-PCR for Patient 1 and 2. Fold Change = miRNA expression in adenocarcinoma tissue normalized to normal colon tissue expression. CT method normalizing to GAPDH was used to calculate qRTPCR fold change values A) B) A) Hierarchical clustering of fold change in miRNA expression between normal colon and adenocarcinoma tumor samples in two patients. Upregulated features are represented in red and downregulatated features in green. Each column represents a single data set from 1 of 4 total RNA samples. Arrays were set up in a loop design, data were background corrected and latin squares normalized. Data from each tissue are identified below each column. B) Statistically significant changes in novel miRNA sequences (shown in grey boxes) across each patient. Significance is defined as greater than 2-fold change from normal with a p-value < Statistical significance determined by NCode TM profiler software comparing observed variance to model of variance in 10,000 bootstraps. Statistically Significant Array Features A.) B.) P-value < 0.05 Fold Change > 2.0 P-value < 0.05 Fold Change > 2.0 DownregulatedUpregulated