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BIOO LIFE SCIENCE PRODUCTS In Vivo RNA-LANCER II Manual Catalog #:3410-01 BIOO Scientific

DO NOT use this protocol to run your assays.

1 BIOO LIFE SCIENCE PRODUCTS In Vivo RNA-LANCER II Manual Catalog #: BIOO Scientific Corp FOR REFERENCE PURPOSES. This manual is for Reference Purposes Only. DO NOT use this protocol to run your assays. Periodically, optimizations and revisions are made to the kit and protocol, so it is important to always use the protocol included with the kit.

2 GENERAL INFORMATION Product Description The MaxSuppressor In Vivo RNA-LANCEr II is a patent-pending, proprietary formulation composed of neutral lipid, non-ionic detergent, oil and small molecules which enable highly efficient delivery of RNAi agents into animals. This novel product has been shown to be more robust and efficient at penetrating tissues and organs than other delivery agents. When manufacturing this reagent, all components of the formulation are dissolved and mixed in an organic solvent to obtain a homogeneous mixture. The intent is to obtain a clear soluble suspension of the components. Once the components are mixed, the organic solvent is Syringes for injections (20) Micro-Homogenizers (2) C C completely removed to yield a semi-dry solution which is supplied to the customer in 4 glass vials in the kit. Each vial has enough material for 5 injections (20 injections total). In addition to the 4 vials of MaxSuppressor In Vivo RNA-LANCEr II, the kit contains MaxSuppressor Microhomogenizers, for tissue disruption, all necessary buffers and sterile syringes. The shelf life of the kit is 12 months at -20ºC before RNAi agent is added to the vial and 3 months at -80ºC once the RNAi agent has been added to the vial. Required Materials Not Provided 10, 20, 200 and 1000 L pipettes RNase-Free pipette tips Lipid Extruder or Sonicator Warnings and Precautions Kit Contents Storage RNA-LANCEr II -20 C (4 vials semi-dry solution, 50 µl) Sterile RNase-Free Water 4 C (10 ml) Sterile RNase-Free 10X PBS 4 C (10 ml) BIOO strongly recommends that you read the following warnings and precautions. Periodically, optimizations and revisions are made to the components and manual. Therefore, it is important to follow the protocol included with the kit. If you need further assistance, you may contact your local distributor or BIOO at rnai@biooscientific.com. Do not use the kit past the expiration date. Try to maintain a laboratory temperature of 20º 25ºC (68º 77ºF). Make sure you are using only the sterile, RNase-free water that is provided with the kit, as water quality is very important. Caution should be taken when working with animals. Always wear chemical-resistant gloves, safety goggles, and other protective clothing when handling animals. BIOO LIFE SCIENCE PRODUCTS 1

3 Procedure Overview The procedure to package RNAi agents into the phospholipid-oil emulsion takes less than 1-hour. To use MaxSuppressor In Vivo RNA-LANCEr II simply mix 110 g of your RNAi agent with a single tube of semi-dry MaxSuppressor In Vivo RNA-LANCEr II agent in total volume based on injection volume desired, in 1X PBS; extrude using the Lipid Extruder (Bioo Scientific cat. #: ) with the supplied 100 nm pore sizing filters (or sonicate) and then directly inject into the animal (see Workflow diagram below). MAXSUPPRESSOR In Vivo RNA-LANCEr-II Workflow BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product. The MaxSuppressor TM IN VIVO RNA-LANCER II is intended for laboratory use only, unless otherwise indicated. This product is NOT for therapeutic use. MaxSuppressor is a Trademark of Bioo Scientific Corporation (BIOO). BIOO LIFE SCIENCE PRODUCTS 2

GENERAL PROTOCOL FOR THE PREPARATION OF RNAi AGENTS 1. Using the chart below, determine the final injection volume, based on the delivery route to be used.

3 and 4. 2. Add 100 g of RNAi agent plus 10% overage into a single glass tube of MaxSuppresor In Vivo RNA-LANCEr. For low-volume injections (<100 L), use a concentrated RNAi stock (10-20 mg/ml).

4 GENERAL PROTOCOL FOR THE PREPARATION OF RNAi AGENTS 1. Using the chart below, determine the final injection volume, based on the delivery route to be used. NOTE 1: The semi-dried formulation is composed of a volume of 50 L of hydrophobic reagents and small molecules which should be taken into account during steps 2, 3 and Add 100 g of RNAi agent plus 10% overage into a single glass tube of MaxSuppresor In Vivo RNA-LANCEr. For low-volume injections (<100 L), use a concentrated RNAi stock (10-20 mg/ml). NOTE 2: Changing the amount of RNAi agent to greater or less than 100 g will influence the delivery efficiency. 3. Based upon what the final volume will be, add the appropriate volume of Sterile RNase- free 10X PBS to make the solution 1X PBS. BIOO LIFE SCIENCE PRODUCTS 3

4. Add Sterile RNase-free water, if needed. For example: 5. Disperse the liposome emulsion in aqueous solution using either the Lipid Extruder (Bioo Scientific cat.

5 4. Add Sterile RNase-free water, if needed. For example: 5. Disperse the liposome emulsion in aqueous solution using either the Lipid Extruder (Bioo Scientific cat. # ) or a water bath sonicator. Generally, sonication is carried out by immersing the formulation vial into the water bath sonicator for 5 minutes on a high power setting. NOTE 3: Extrusion increases efficiency of encapsulation and sizes liposomes to 100 nm. Sonication sizes liposomes to nm. 6. Inject the total volume of the RNAi agent/phospholipid-oil emulsion into animal using the desired route of administration. For additional information on the routes of delivery and specific protocols see the Routes of Delivery section below. NOTE 4: For rats, the total volume of injected material should not exceed 0.5 ml of solution into the tail vein. However, because the weight of rats can range from 100 g to 800 g, larger amounts, and custom formulations of MaxSuppressor In Vivo RNA-LANCEr can be obtained (please inquire for details). BIOO LIFE SCIENCE PRODUCTS 4

6 PROTOCOLS FOR IN VIVO INJECTION OF RNAi AGENTS RNAi agents can be administered using many methods. For example, the delivery route can be focused so that more efficient uptake and reduction may be obtained in your desired destination. The RNAi agent may also be directly injected into a subcutaneous tumor or into a specific organ using surgical procedures. ROUTES OF DELIVERY Systemic Delivery Routes: Intravenous (IV) Intraperitoneal (IP) Subcutaneous (Sub-Q) Low Pressure Tail Vein (LPTV) Local Delivery Routes: Inhalation Intraocular Intrathecal (spinal canal) *Animal care protocols Studies using MaxSuppressor In Vivo RNA-LANCEr II are commonly used and approved by IACUC. BIOO LIFE SCIENCE PRODUCTS 5

7 IV Intravenous Injection 1. Warm the mouse tail to about 37ºC (approximately 10 minutes) using a heat lamp. 2. The mouse should be restrained using a mouse restraining device or by hand by trained personnel. 3. Disinfect the tail using an alcohol swab and slightly rotate the tail to visualize the vein. 4. Once the vein has been located, disinfect the site of injection and insert the needle at slight angle. Inject slowly (~20 L/sec) and watch for clearing of the blood in the vein. If a bulge appears in the tail, remove the needle and repeat the process proximal to the previous site. 5. Upon completion remove needle and apply pressure to the injection site. IP Intraperitoneal Injection 1. Restrain the mouse using the fold of skin near the neck and expose the abdomen. 2. Disinfect the injection site by swabbing the area with an alcohol swab. 3. Insert the needle, at an angle, into the peritoneal cavity of the abdomen, avoiding puncture of internal organs, and inject slowly (20 L/sec). IN Intranasal Administration 1. Mice should be anesthetized by injecting ml of anesthetic, such as ketamine or Phenobarbital in the lower flank of the mouse. 2. Place the mouse on its back over a warm pad or under a heat lamp for about 10 minutes. 3. Inject µl of the RNA agent formulation in each nostril using a narrow pipet tip. Inject slowly and wait about 1 minute between injections to help recovery. IT Intratumoral Injection 1. When the subcutaneous tumor areas reach at least mm 3 (approx days post injection) in size, the mice are ready for injection. Larger tumors may also be targeted. The volume of injection is µl/tumor, depending on the tumor size. 2. The mouse should be restrained using a mouse restraining device or by hand. 3. Use forceps to gently hold the tumor. 4. Disinfect the site of injection and insert the needle directly into the tumor, and penetrate as deeply as possible without passing through the tumor. 5. Slowly push about µl into the tumor. Then gently retract the needle and shift to a different location within the tumor and inject an additional µl of preparation. Repeat this process until all material has been injected. 6. After injection, leave the needle in the tumor for about 20 seconds, and then slowly pull out and pinch the opening with fine forceps to avoid leakage. BIOO LIFE SCIENCE PRODUCTS 6

8 After injection of the RNAi agent the recommended incubation time in the animal is 3-4 days. Sacrifice the animal using an approved method and then harvest the desired tissues. Be sure that the samples are properly stored. In general, samples should be refrigerated at 2-4ºC for no more than 1-2 days. If samples need to be stored for longer than 2 days, freeze them to -80ºC. Frozen samples can be thawed at room temperature (20 25ºC/68 77ºF) or in a refrigerator before use. Serum 1. Blood should be collected without anticoagulant and left at room temperature for 3 hours or at 4 C overnight to clot. 2. Centrifuge at 4 C, 10 minutes, 3,000 x g. 3. Take the serum from the upper layer of the blood sample. 4. Use the serum directly in desired assay. Tissue-Protein Extraction 1. Add 500 µl of Protein Extraction Buffer (Bioo Scientific cat. # ) to 500 mg or less of tissue samples. 2. Grind sample using Micro-Homogenizer (Bioo Scientific cat. # ) or pestle. 3. Freeze sample for 30 minutes -80 C freezer or a dry ice/ethanol bath. 4. Thaw sample at room temperature (20-25 C / F) and spin in microcentrifuge at 14,000 rpm for 10 minutes. 5. Remove supernatant, dispose of pellet. 6. Dilute sample in Bradford Diluent, provided with the MaxDiscovery Bradford Assay Kit (Bioo Scientific cat: ). Recommended starting dilutions are 1:50 and 1: Quantify total protein using MaxDiscovery Bradford Assay Kit. 8. Total concentration of protein in sample should be normalized using Sample Diluent to mg/ml. 9. Use normalized sample in desired assay. Tissue-RNA Extraction ANIMAL HARVEST AND SAMPLE PREPARATION We recommend using BiooPure RNA Isolation Reagent (Bioo Scientific cat. # 5301) to isolate RNA from tissues. BiooPure is a single-phase reagent for extraction of total RNA or enriched small RNA (including mi RNA) from solid tissues, cultured cells, and cell-free fluids such as serum and plasma. The BiooPure reagent contains guanidinium, a powerful chaotropic agent effective for rapidly inactivating nucleases, and phenol, an organic solvent used to denature and separate proteins and DNA from RNA. BIOO LIFE SCIENCE PRODUCTS 7

9 Solid Tissue 1. Manually grind or mechanically disrupt tissue to form a smooth homogeneous lysate with no particles of intact tissue, using approximately 10 volumes of BiooPure reagent per mass amount of tissue. For example use 1 ml of BiooPure reagent per 100 milligrams (mg) of solid tissue. If weighing the tissue is inconvenient or impractical, the volume of BiooPure reagent to use may be estimated. For ease in processing tissue samples, at least 1 ml of BiooPure Reagent per prep is recommended. For example, use 1 ml of BiooPure for 50 mg of tissue. For processing solid tissue that has been stored in Ambion s RNALater preservative, remove excess RNALater by blotting and lightly pressing the tissue on an absorbent surface. 2. Transfer the tissue lysate to a microfuge tube or larger tube as needed. Tissue lysates may be stored at -20 C until ready to proceed further. In general, RNA is stable in lysates stored at -20 C for several months. Cell-Free Fluids (Blood serum or plasma) 1. Use 3-4 volumes of BiooPure reagent per volume of sample. 2. Thoroughly mix the sample with BiooPure reagent by vortexing or shaking. Note: The ratio of BiooPure reagent to cell-free fluid can be adjusted empirically according to sample type. For preps that will be carried out in microfuge tubes, using a 4:1 ratio of sample:bioopure reagent, the maximum volume of sample is ~250 µl per 1.5 ml tube or ~330 µl per 2 ml tube; this will allow sufficient volume to add BCP and vortex the sample thoroughly in the next step. Sample lysates in BiooPure reagent may be stored at -20 C until ready to proceed further. In general, RNA is stable in lysates stored at -20 C for at least several months. Related Bioo Scientific Products Cat # Lipid extruder Extruder membranes (100nm) NextFect RNA Transfection Reagent NextFect DNA Transfection Reagent NextExpress Protein Expression Kit T3 - Max Conjugation kit NeuroAim In Vitro Transduction Reagent MyeloAim In Vitro Transduction Reagent huleukoaim In Vitro Transduction Reagent muleukoaim In Vitro Transduction Reagent MaxCarrier HER2 Antibody Conjugate HER2 monoclonal antibbody RNase-Free Sterile Saline RNase-Free Sterile Ringers , , , , , , , , , , , , , , , , , , BIOO LIFE SCIENCE PRODUCTS 8

10 SELECTED REFERENCES Ben-Shushan, D, et al. (2013) Overcoming obstacles in microrna delivery towards improved cancer therapy. Drug Deliv. and Transl. Res. doi /s De Vito C, et al. (2011) Let-7a Is a Direct EWS-FLI-1 Target Implicated in Ewing s Sarcoma Development. PLoS ONE 6(8): e doi: /journal.pone De Vito C, et al. (2012) A TARBP2-Dependent mirna Expression Profile Underlies Cancer Stem Cell Properties and Provides Candidate Therapeutic Reagents in Ewing Sarcoma. Cancer Cell. DOI /j.ccr Imam JS, Plyler JR, Bansal H, Prajapati S, Bansal S, et al. (2012) Genomic Loss of Tumor Suppressor mirna-204 Promotes Cancer Cell Migration and Invasion by Activating AKT/mTOR/Rac1 Signaling and Actin Reorganization. PLoS ONE 7(12): e doi: /journal.pone Liu, C. et al. (January 16, 2011) The microrna mir-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. Nature Medicine doi: /nm.2284 Letter. Luna C, Li G, Huang J, Qiu J, Wu J, et al. (2012) Regulation of Trabecular Meshwork Cell Contraction and Intraocular Pressure by mir-200c. PLoS ONE 7(12): e doi: /journal.pone Sun, CY., She XM., et al. (Oct 26, 2012) mir-15a and mir-16 affect the angiogenesis of multiple myeloma by targeting VEGF. Carcinogenesis. Trang, P. et al. (March 22, 2011) Systemic Delivery of Tumor Suppressor microrna Mimics Using a Neutral Lipid Emulsion Inhibits Lung Tumors in Mice. Molecular Therapy doi: /mt Wang, L. et al. (2012) Loss of mir-29 in Myoblasts Contributes to Dystrophic Muscle Pathogenesis. Molecular Therapy doi: /mt Wiggins, J.F. et al. (July 15, 2010) Development of a Lung Cancer Therapeutic Based on the Tumor Suppressor MicroRNA-34. Cancer Res. 70: Wu, Y. et al. (2011) MicroRNA Delivery by Cationic Lipoplexes for Lung Cancer Therapy. Mol. Pharmaceutics. 8, BIOO LIFE SCIENCE PRODUCTS 9

11 SAMPLE RESULTS Figure 1 - The Bioo-formulated sirna (IN-BFR) perform significantly better than naked or sirna formulated with a competitors formulation: BIOO LIFE SCIENCE PRODUCTS 10

Figure 2 - The relative GAPDH protein levels in the organs of (A) mice or (B) rats

The GAPDH levels are compared to animals treated with a negative control sirna.

208.2246 Fax: (512) 707-8122 Made in USA BIOO Research Products Group

12 Figure 2 - The relative GAPDH protein levels in the organs of (A) mice or (B) rats injected with MaxSuppressor In Vivo RNA- LANCEr II containing GAPDH sirna. The GAPDH levels are compared to animals treated with a negative control sirna. Bioo Scientific Corporation 3913 Todd Lane Suite 312 Austin, TX USA Tel: Fax: (512) Made in USA BIOO Research Products Group info@biooscientific.com techsupport5@biooscientific.com BIOO LIFE SCIENCE PRODUCTS 11

BIOO RESEARCH PRODUCTS. Alkaline Phosphatase (AP) Color Endpoint Assay Kit Manual Catalog #:

BIOO RESEARCH PRODUCTS. Alkaline Phosphatase (AP) Color Endpoint Assay Kit Manual Catalog #: BIOO RESEARCH PRODUCTS Alkaline Phosphatase (AP) Color Endpoint Assay Kit Manual Catalog #: 3460-09 BIOO Scientific Corp. 2011 TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Description... 1 Procedure