SUPER SENSITIVE TM. mirna in FFPE. Next-Gen in situ Tissue Signature Markers Potential tool for Characterization of CUP

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1 SUPER SENSITIVE TM mirn in FFPE Next-Gen in situ Tissue Signature Markers Potential tool for haracterization of UP

2 mirn in FFPE mirn Processing MicroRNs (mirns) are endogenous, non-coding RNs known to regulate gene expression by translational repression or RN cleavage. The figure below shows the steps in which the mirn genes are processed into the RN induced silencing complex (RIS). Since mirn has been observed to deregulate during progression of different cancer stages from normal to malignant and metastasis, the expression profile as a result of this deregulation can be exploited as a potential biomarker for cancer characterization. Nucleus ytoplasm mirn genes RN Pol II or III Drosha/ DGR8 Pre-miRN Exportin 5 Pre-miRN Pri-miRN DIER RIS complex Unwind mirn duplex mirn mirn* mrn Targeting Degradation mirn iogenesis iogenex MicroRN Probes New Potential Molecular Tool for haracterization of carcinoma of unknown primary (UP) lassification of cancer subtypes utomated Protocols Optimized for automated ISH staining by Xmatrx ELITE Ready-to-use reagents for FFPE tissues Highly Specific and Sensitive Probes Proprietary technology for clean intense stains in situ context of tissue morphology Manual Protocols Optimized for standardized manual ISH staining Ready-to-use reagents for FFPE tissues

3 Role of mirn in Gene Regulation mirn regulates gene expression either by blocking the translation or destabilizing the targeted mrn transcripts. mirn as part of RIS complex targets the mrn to inhibit its translation thus brings about the necessary regulatory control. On the other hand mirn- RIS complex binds to 3 -UTRs of mrn to induce mrn degradation or destabilization by decapping and de-adenylation of mrn there by inhibiting translation. The targeted mrn along with the mirn-ris complex is often sequestered to P-bodies for further regulation by degradation or subsequent use. Translation block at initiation step (a) Ribosome Translation repression mirnp/ris (b) mirnp/ris Translation repression Translation block at elongation step Ribosome run-off and aggregation Ribosome mirnp/ris (c) Nascent polypoptide chains protease Polypeptide Proteolysis mirnp/ris P-body Decay mirn Storage mirnp (d) GW182 mirnp/ris cr4:not1 Deadenylation mirn degradation mrn exit and reuse mrn decay Translation regulated by mirn mirn Potential linical pplications arcinoma of unknown primary (UP) accounts for an estimated 3% to 5% of all metastatic cancers and one of the ten most frequent cancer diagnosed worldwide. Recent studies on tissue specific tumor markers indicated that mirn expression is correlated with tumor signature and mirn is frequently regulated in cancer. This fact indicated a differential expression pattern of mirn in normal, neoplastic, pre-malignant and malignant tissues. Therefore, in situ detection of mirn differential expression in different stages of cancer makes the expression profiling a potential tool not only for cancer characterization but also for accurate UP characterization and disease management.

4 High Specificity and Sensitivity Specific Intense Stain The cervical cancer tissues were stained using scramble, mir-146a, and mutant mir-146a probes. Mutant mir-146a probe has 3 different nucleotides compared with normal mir-146a probe.. mir-146a (positive). mutant mir-146a. Scramble Probe mirn Knockout Tissues mir-146a staining was observed only in wild type mouse spleen but not in 146a knockout mouse spleen tissues. lue color is the signal from chromogen IP and P-anti-fluorescein antibody.. mir-146a WT spleen. mir-146a knockout spleen mirn Positive ontrol Probe We provide pre-labeled snrn U6 probe as the positive control, which is a good tool to optimize the sensitvity and performance of the mirn detection protocol initially in particular tissue.. snrn U6

5 More mirns... Hsa-miR-145 Hsa-miR-145, a novel smooth muscle cell phenotypic marker, is the most abundant mirn in vascular wall of multiple tissues, such as bladder, stomach and small intestine.. mir-145. mutant mir-145. Scramble Probe Hsa-miR-1 Hsa-miR-1 is believed to be specifically expressed in adult cardiac and skeletal muscle tissues. It plays a key role in the development and differentiation of smooth and skeletal muscles. mir-1 has been found down-regulated in lung, colon, and prostate cancer.. mir-1 Skeletal Muscle. mir-1 Mutant probe. Scramble Probe Hsa-miR-205. a reast mir-205. a Lung mir-205. Scramble Probe (a Lung)

6 More mirns... D F E. mir-328 in Tonsil. mir-205 in Human Lung a.. mir-155 in Human Spleen D. mir-1 Human Skeletal Muscle E. mir -145 in Human Small Intestine F. mir-222 in Human ervical a. Elegance on the Glass Slide......from Microtome to Microscope Xmatrx ELITE assures accurate reagent dispensing using liquid level sensor, even distribution of reagents on the specimen and up to 75% reduced reagent consumption via coverslip micro-chamber, precise temperature control on each slide by ext TM and eliminates cross contamination through the use of disposable pipette tips. ontact Information: Xmatrx ELITE maximizes the testing capacity, minimizes hands-on time, reduces errors and produces consistent and accurate results. This under-scores our commitment to provide a system to meet the needs of molecular pathology laboratory of today, tomorrow and beyond iogenex Laboratories, Inc. ll Rights Reserved US: Milmont Drive, Fremont T: , US F: India: 225 herlapally, Industrial Development rea, Phase II T: Hyderabad, ndhra Pradesh, India F: /21 hina: F, Jingjiang Xiangyang Tower T: Nanjing Xi Road (W), Shanghai, , hina F: support@biogenex.com