Functional Analysis. LNA longrna GapmeR

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1 Functional Analysis LNA longrna GapmeR Instruction manual v2.0 August 2013

2 Table of contents Product Summary Content Additional required materials Product description Applications Shipping and storage Protocol Resuspension Transfection guidelines Unassisted uptake (Gymnosis) Related products References

3 Product summary Content LNA longrna GapmeR Product No* Product description Label Amount supplied LNA longrna GapmeR in vitro Standard Ready-to-label XX LNA longrna GapmeR in vitro Premium XX LNA longrna GapmeR in vivo Ready XX LNA longrna GapmeR in vivo Ready 20 nmol of nucleotide, *Product No suffix digits varies depending on the type of label applied. Negative controls designed for use in with the above gene specific LNA longrna GapmeR products Product No* Product description Label Amount supplied Negative control A, in vitro Standard Ready-to-label XX Negative control A, in vitro Premium XX Negative control A, in vivo Ready XX Negative control A, in vivo Ready 20 nmol of nucleotide, Negative control B, in vitro Standard Ready-to-label XX Negative control B, in vitro Premium XX Negative control B, in vivo Ready XX Negative control B, in vivo Ready 20 nmol of nucleotide, *Product No suffix digits varies depending on the type of label applied. 3

4 Additional required materials Nuclease-free water Microcentrifuge DNase-free microcentrifuge tubes or microtiter plate Cell culture plates Cell culture medium Transfection reagent Product description LNA longrna GapmeRs are antisense oligonucleotides with perfect sequence complementary to their RNA target. When introduced into cells, they sequester their target RNA in highly stable DNA: RNA heteroduplexes, leading to RNase H mediated target degradation. The sequences of the oligonucleotides and their LNA spiking patterns have been carefully designed by Exiqon s GapmeR Design Algorithm to achieve high target affinity with excellent sequence specificity and biological stability, while keeping the self-annealing properties to a minimum. Applications LNA longrna GapmeRs are potent antisense oligonucleotides primarily used to study the functions of mrna or long noncoding RNA by assessing the biological consequences of inhibiting their expression. The effect of silencing an mrna or long non coding RNA can be studied in numerous ways, such as using cellular assays to monitor cell proliferation, cell differentiation, or apoptosis. The effect on gene expression can also be measured at the level of RNA or putative protein targets. 4

5 Shipping and storage This product is shipped dried down at room temperature. The unopened vial should be stored at -20 C or below. Fluorescence-labeled oligonucleotides should be protected from light to avoid bleaching. Shelf life is at least 6 months after shipping date when stored in this manner. Exposure to higher ambient temperatures during shipment does not pose any risk to the stability of the oligonucleotides. Oligonucleotides are degraded by repeated freeze-thaw cycles, especially when in solution. After resuspension, it is recommended to aliquot the product before storage. For storage at -20 C, please use a constant temperature freezer. Important note Do not store in frost-free freezer with automatic thaw-freeze cycles. 5

6 Protocol Important note LNA oligonucleotides are susceptible to degradation by exogenous nucleases introduced during handling. Wear powder-free gloves when handling this product. Use DNase-free reagents and filter pipette tips. Whenever possible, work under a tissue culture hood. Resuspension 6 Step 1 Briefly centrifuge the screw cap vial at low speed (maximum 4,000 x g) to make sure that all material is collected at the bottom of the wells before removing the cap in step 2. Step 2 Remove screw cap carefully. Step 3 Add nuclease-free, sterile water using a pipette with a sterile filter tip to achieve the desired concentration. Stock solutions should not be lower than 10 μm (Adding 100 μl water to 5 nmole LNA longrna GapmeR will yield a 50 μm solution). Step 4 Let the vial stand for a few minutes at ambient temperature. Step 5 Gently pipette up and down 5 times to resuspend. Step 6 Repeat steps 4 and 5. Step 7 We recommend aliquoting the gapmer solution into sister tubes to limit the number of thaw-freeze cycles.

7 Step 8 Store at 20 C. Step 9 Avoid thaw-freezing more than 5 times (working solutions can be stored at 4 C for a period of maximum 14 days). Transfection guidelines Transfection efficiency varies according to cell type and the transfection reagent used. The optimal combination of cell type, transfection reagent and transfection conditions must be determined empirically. Optimizing transfection efficiencies is crucial for maximizing mrna or long non-coding RNA inhibition while minimizing secondary effects. Expect to spend some time finding the optimal transfection conditions. Optimal transfection conditions are found by identifying efficient transfection reagents for each cell line and by adjusting: Amount of transfection reagent Amount of LNA longrna GapmeR Cell density at time of transfection Order of transfection (plating cells before transfection or plating cells at the moment of transfection) Length of exposure of cells to transfection reagent/oligonucleotide complex Most protocols recommend maintaining mammalian cells in the medium used for transfection for 24 hours. The transfection medium should then be replaced with fresh medium to maximize viability of the cell culture. Normally LNA longrna GapmeR displays potent activity at final concentrations of 1-50 nm, but a more extensive range of nm can be analyzed in optimization experiments. Always remember to perform adequate controls to ensure that the resulting phenotype is due to antisense inhibition of the targeted RNA. 7

8 Table 1. Cell culture plate 96 well 24 well 12 well 6 well Transfection reagent A) μl 1 3 μl 2 4 μl 3 36 μl LNA longrna GapmeR B) 5 pmole 25 pmole 50 pmole 150 pmole Cell density (cells/well) C) Final volume per well 100 μl 500 μl 1000 μl 3000 μl A) B) C) Refer to the instructions provided by the transfection reagent supplier. The amount shown yields a LNA longrna GapmeR concentration of 50 nm. Optimal cell density varies with the cell type depending on cell size and growth characteristics. In general, 30 70% confluency is recommended. Unassisted uptake (Gymnosis) LNA longrna GapmeR can also be introduced into cells unassisted by any transfection reagents. LNA antisense gapmers have been shown to have high potential in penetrating the cell-membrane. It has been demonstrated that LNA antisense gapmers can be taken up into the cell unassisted (the term gymnosis was coined for this process, which denotes naked delivery) (Stein, et al. 2010, Soifer, et al. 2012). This method is especially useful with cells that are notoriously difficult to transfect (e.g. non-adherent cells or primary cells). This protocol should be applicable to most mammalian cell lines. However, cellular confluency, working oligo concentrations, and the time of incubation may have to be optimized for different cell lines of interest. It is also important that oligo-containing culture media should be left in the wells for the whole duration of treatment. Always remember to perform adequate controls to ensure that the resulting phenotype is due to antisense inhibition of the targeted RNA. Optimal gymnotic conditions are determined by adjusting the following parameters: Amount of LNA longrna GapmeR. Appropriate LNA longrna GapmeR solution should be prepared at the final concentration (1-50 μm) in the appropriate fresh culture media before introducing to the cells Cell density. To avoid problems associated with over-confluency of cells, plating cell density can be appropriately decreased if long period of incubation is anticipated Length of exposure of cells to LNA longrna GapmeR. A general guideline for the incubation time would be 1 to 6 days after initial treatment. If longer incubation is required (more than 6 days), it is recommended that media is replaced with fresh media containing LNA longrna GapmeR 8

9 Related products Exiqon offers several pre-designed and custom products enabling new discoveries concerning the isolation, expression analysis, function and spatial distribution of mrnas and lncrnas: Custom LNA longrna Gapmer for in vivo use (custom large scale) The same high quality and purity as the in vivo Ready GapmeRs available with custom large scale synthesis and modifications. Please contact us for more details. Custom LNA mrna Detection Probes For in situ hybridization of mrna and long non-coding RNA. Use Exiqon s Custom LNA mrna Detection Probe design tool to design highly sensitive and specific LNA probes with a variety of labels. See for more details. mircury RNA Isolation Kits Prepare high-quality total RNA from a wide range of organisms and tissues using fast, phenol-free protocols. See for details. Custom LNA qpcr Design custom LNA -enhanced primer sets for mrna and lncrna. Use our web tool to design high performance primers compatible with Exiqon s Universal cdna Synthesis and ExiLENT SYBR Green Master Mix kits. See for more details. Custom LNA oligonucleotides LNA oligonucleotides can be successfully used in a wide range of RNA applications. Design your own using our online tools or contact us for assistance. See for details. 9

10 References Stein CA, Hansen JB, Lai J, et al., Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents, Nucleic Acids Research 38(1): e3, 2010 Soifer HS, Koch T, Lai J, et al., Silencing of gene expression by gymnotic delivery of antisense oligonucleotides, Methods Mol Biol. 815:333, Literature citations: Please refer to LNA longrna GapmeR when describing a procedure for publication using this product. Patents and Trademarks Exiqon, LNA and mircury are registered trademarks of Exiqon A/S, Vedbaek, Denmark. SYBR Green is a licensed trademark of Invitrogen. All other trademarks are the property of their respective owners. Locked-nucleic Acids (LNAs ) are protected by US Pat No. 6,268,490, US Pat No. 6,770,748, US Pat No. 6,639,059, US Pat No. 6,734,291 and other applications and patents owned or licensed by Exiqon A/S. Products are provided to buyers for research use only. The products in their original or any modified form may be used only for the buyer s internal research purposes and not for commercial, diagnostic, therapeutic, or other use, including contract research. The buyer may not provide products to third parties in their original or any modified form. The purchase of products does not include or carry an implied right or license for the buyer to use such products in their original or any modified form in the provision of services to third parties, and a license must be obtained directly from Exiqon A/S for such uses. For research use only. Not for use in diagnostic procedures. Copyright 2013 Exiqon. All rights reserved. 10

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12 Outside North America Exiqon A/S Skelstedet 16 DK-2950 Vedbaek Denmark Phone Fax North America Exiqon Inc. 12 Gill Street, Suite 1650 Woburn, MA United States Phone (781) Fax (781) v2.0-08/2013 exiqon.com