Anti-miR鈩? miRNA Inhibitor Library Product Information Sheet
Product: Anti-miR鈩? miRNA Inhibitor Library
Catalog #: 17200V2
Lot #: AMO013U8
Amount: 0.25 nmol each Anti-miR miRNA Inhibitor
Content: 328 Anti-miR miRNA Inhibitors targeting human miRNAs in miRBase Sequence Database Version 8.0
Total of 4 96-well plates (plates are Axygen #PCR96FS; www.axygen.com)
3 plates each with 88 Anti-miR miRNA Inhibitors
1 plate with 64 Anti-miR miRNA Inhibitors
See accompanying CD for target miRNA sequence information and additional Anti-miR Inhibitor details.
Purity: HPLC purified
Appearance: Powder
Storage: Store at 鈥?20掳C or 鈥?80藲C. Do not store in a frost-free freezer. (Dried oligonucleotides are shipped at
ambient temperature.)
Shelf Life: 1 year, when stored at 鈥?20掳C or 鈥?80藲C.
QUALITY CONTROL
Identity: A MALDI-TOF mass spectrometer is used to identify the correct mass of the single-stranded RNA
oligonucleotides.
Purity: Analytical HPLC of the final purified single-stranded RNA oligonucleotides is used to confirm >90% purity.
USER INFORMATION
General Information: Anti-miR鈩? miRNA Inhibitors (patent pending) are designed to bind to and inhibit the activity of endogenous
miRNAs when introduced into cells. These RNA-based inhibitors are chemically modified to increase their
stability and to improve their activity.
Anti-miR miRNA Inhibitors can be delivered to mammalian cells by chemical transfection or electroporation.
Handling Instructions: RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during
handling. As a precaution, do not handle this product with ungloved hands. RNase-free reagents, barrier
pipette tips, and tubes should be used. Upon receipt, the library may be safely stored in a non-frost-free
freezer for 1 year at 鈥?20掳C or 鈥?80藲C (dried oligonucleotides are shipped at ambient temperature).
Resuspension of siRNAs
Centrifuge each plate at low speed (maximum RCF 4,000 X g) to collect the contents at the bottom of the
wells before removing the seal in step 1.
Important: Perform this process under a tissue culture hood.
1. Remove seal carefully.
2. Add nuclease-free, sterile water using a multichannel pipette and sterile tips to achieve the desired
concentration.* (Adding 50 碌L water to 0.25 nmol Anti-miR Inhibitor will make a 5 碌M stock.)
3. Gently pipette up and down 5 times to resuspend.
4. (Optional) Aliquot the Anti-miR Inhibitors into one or more daughter plates, to limit the number of
freeze-thaw cycles to which the Pre-miR Inhibitors are subjected.
5. Place a new sterile seal (such as Axygen PCR-AS-200) on the plate before storing.
6. Store at 鈥?80掳C until ready to use.
* Ambion provides an online calculator for suspension of dried oligonucleotides on its website at
http://www.ambion.com/techlib/append/oligo_dilution.html
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Anti-miR鈩? miRNA Inhibitor Library Cat #AM17200V2
Applications: The key application of Anti-miR miRNA Inhibitors is to suppress endogenous miRNA function (1, 2). For this
application, an Anti-miR miRNA Inhibitor is introduced into cells that express the corresponding miRNA, and
expression of the endogenous target of the corresponding miRNA, or of a reporter construct containing the
predicted target miRNA binding site, is measured.
Suppression of Endogenous miRNA Function Using an miRNA Target Reporter Plasmid
Reporter plasmids with an miRNA binding site in the 3鈥? UTR of the reporter gene, to provide a target for
miRNA activity, are commonly used to analyze miRNA function on predicted miRNA binding sites. When
cotransfected with a reporter plasmid containing the associated miRNA binding site, an Anti-miR miRNA
Inhibitor can be assessed for its ability to relieve inhibition of gene expression caused by an endogenous
miRNA. This type of experiment typically also includes a second type of reporter plasmid to normalize for
transfection variation.
Transfection Procedure: We routinely transfect Anti-miR miRNA Inhibitors at a final concentration of
15鈥?100 nM and reporter vector DNA (such as pMIR-REPORT鈩?, Cat #5795) at 200鈥?800 ng using siPORT鈩?
NeoFX鈩? Transfection Agent, and assay the expression of the reporter gene 24鈥?48 hr later. Optimal levels
of transfection agent must be determined empirically. A pMIR-REPORT 尾-gal Control Plasmid, included in
the pMIR-REPORT kit, can be used for normalization of transfection efficiency. See the kit Instruction
Manual for a detailed protocol, available at:
http://www.ambion.com/catalog/CatNum.php?5795
Suppression of Endogenous miRNA Function Using Endogenous miRNA Targets
To analyze the effects that miRNAs have on a biological process or on an endogenous target, an Anti-miR
miRNA Inhibitor can be transfected into cells to evaluate whether the miRNA effect is reversed.
Transfection Procedure: Transfection efficiency varies according to cell type and the transfection agent
used; we suggest testing Anti-miR miRNA Inhibitors at a final concentration of 15鈥?100 nM.
Transfection of Mammalian Cells with Anti-miR miRNA Inhibitors
As with other small nucleic acids, such as siRNAs and antisense oligonucleotides, the efficiency with which
mammalian cells are transfected with Anti-miR miRNA Inhibitors will vary according to cell type and the
transfection agent used. The optimal concentration used for transfections should be determined empirically.
We have found that Anti-miR Inhibitors typically work best when transfected at a final concentration of
15鈥?100 nM. However, a more extensive concentration range from 1鈥?100 nM can be analyzed in optimization
experiments.The following chart provides general starting points for transfection of Anti-miR Precursors into
cultured mammalian cells.
General Transfection Starting Points for Mammalian Cells
96 well 24 well 12 well
a
0.3鈥?1.0 碌L 1鈥?3 碌L 2鈥?4 碌L
Transfection Agent
b
3 pmol 15 pmol 30 pmol
Anti-miR Inhibitor
c
6000 cells/well 40,000 cells/well 80,000 cells/well
Cell density
Final volume per well 0.1 mL 0.5 mL 1.0 mL
a
Refer to the instructions provided with your transfection agent for the recommended volume.
b
The amount shown results in a final Anti-miR Inhibitor concentration of 30 nM. The amount of
Anti-miR Inhibitor required for maximal Anti-miR Inhibitor activity will vary from cell type to cell
type. For a 96-well plate, and a 100 碌L final transfection volume, 3 pmol of a 5 碌M Anti-miR
Inhibitor solution is 0.6 碌L. Robotic pipettors may require volumes of 2鈥?5 碌L for accurate
pipetting. To increase pipetting volumes and accuracy, we recommend preparing a plate with
a dilution of your stock Anti-miR Inhibitor when preparing transfection complexes.
c
Optimal cell density will vary from cell type to cell type, depending on cell size and growth
characteristics. In general, 30鈥?70% confluency is recommended.
Transfection Optimization
Optimizing transfection efficiency is crucial for maximizing Anti-miR Inhibitor activity while minimizing
cytoxicity. Optimal transfection efficiencies are achieved by identifying an effective transfection agent for
each cell type and by adjusting (in order of importance):
鈥? Amount of transfection agent
鈥? Amount and type of RNA oligonucleotide
鈥? Cell density at the time of transfection
鈥? Order of transfection (pre-plating cells or plating cells/transfecting in tandem)
鈥? Length of exposure of cells to transfection agent/siRNA complexes
Most protocols recommend maintaining mammalian cells in the medium used for transfection; this avoids
diluting or removing RNA oligonucleotides from the cells by adding medium or washing the cells with new
medium too soon after transfection. We have found that cells typically exhibit greater viability when existing
medium is replaced by fresh medium 24 hours after transfection. Replacing medium after 24 hours
AM17200V2_AMO013U8_1/29/2007
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Anti-miR鈩? miRNA Inhibitor Library Cat #AM17200V2
generally does not appear to change the activity of the transfected Anti-miR miRNA Inhibitor.
Once the conditions for maximal gene silencing are determined, they should be kept constant from
experiment to experiment for a given cell type.
For additional information about small RNA transfection, including transfection conditions for many cell types
and optimization protocols, see Ambion's siRNA Delivery Resource at:
http://www.ambion.com/techlib/resources/delivery
Additional For protocols, background information, a reference, list, and miRNA research tools, see our miRNA
Information: information resource and product guide:
http://www.ambion.com/miRNA
References: 1. Meister G, Landthaler M, Dorsett Y, Tuschl T. (2004) Sequence-specific inhibition of microRNA- and
siRNA-induced RNA silencing. RNA 10(3):544-50.
2. Hutvagner G, Simard MJ, Mello CC, Zamore PD. (2004) Sequence-specific inhibition of small RNA
function. PLoS Biol. 2(4):E98. Epub 2004 Feb 24.
RELATED PRODUCTS
Pre-miR鈩? miRNA Precursor Molecule Library
Cat #AM17300V2
A comprehensive collection of Pre-miR miRNA Precursor Molecules mimicking precursors to human
miRNAs in miRBase Sequence Database Version 8.0.
pMIR-REPORT鈩? miRNA Expression Reporter Vector System
Cat #AM5795
Accurate, quantitative miRNA reporter vector.
siPORT鈩? NeoFX鈩? Transfection Agent
Cat #AM4510 and AM4511
A versatile lipid-based agent for efficient and reproducible transfection of adherent cells while subculturing,
without increased cytotoxicity.
mirVana鈩? PARIS鈩? Kit
Cat #AM1556
Isolate miRNA and proteins from the same sample.
mirVana鈩? Probe and Marker Kit
Cat #AM1554
For end-labeling and purification of small RNA probes and markers.
OTHER INFORMATION
Material Safety Data Sheets (MSDSs) can be printed or downloaded from product-specific links on our website at the
Material Safety Data
following address: www.ambion.com/techlib/msds. Alternatively, e-mail your request to
Sheets:
MSDS_Inquiry_CCRM@appliedbiosystems.com. Specify the catalog or part number(s) of the product(s), and we will e-mail
the associated MSDSs unless you specify a preference for fax delivery. For customers without access to the internet or fax,
our technical service department can fulfill MSDS requests placed by telephone or postal mail. (Requests for postal delivery
require 1鈥?2 weeks for processing.)
For research use only. Not for use in diagnostic procedures.
Warranty and Liability:
Ambion is committed to providing the highest quality reagents at competitive prices. Ambion warrants that for the earlier of
(i) one (1) year from the date of shipment or (ii) until the shelf life date, expiration date, 鈥渦se by鈥? date, "guaranty date" or
other end-of-recommended-use date stated on the product label or in product literature that accompanies shipment of the
product, Ambion's products meet or exceed the performance standards described in the product specification sheets if
stored and used properly. No other warranties of any kind, expressed or implied, are provided by Ambion. WARRANTIES
OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE ARE EXPRESSLY DISCLAIMED. AMBION'S
LIABILITY SHALL NOT EXCEED THE PURCHASE PRICE OF THE PRODUCT. AMBION SHALL HAVE NO LIABILITY
FOR INDIRECT, CONSEQUENTIAL OR INCIDENTAL DAMAGES ARISING FROM THE USE, RESULTS OF USE, OR
INABILITY TO USE ITS PRODUCTS. See the full limited warranty statement that accompanies products for full terms,
conditions and limitations of Ambion's limited product warranty, or contact Ambion for a copy.
Applied Biosystems and AB (Design) are registered trademarks, and Applera is a trademark of Applera Corporation or its
Trademarks, Patents,
subsidiaries in the US and/or certain other countries. Ambion and The RNA Company are registered trademarks and Anti-
and Licensing
miR, mirVana, NeoFX, PARIS, pMIR-REPORT, Pre-miR, and siPORT are trademarks of Ambion, Inc. in the US and/or certain
other countries. All other trademarks are the sole property of their respective owners.
漏 2007 Ambion, Inc., an Applied Biosystems Business. All rights reserved. 4383927A
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