Read MAN0001182_13778.pps_RNAiMAX_20July2012.indd text version

Lipofectamine® RNAiMAX Reagent

Cat. nos. 13778-100 13778-030 13778-075 13778-150 13778-500

Pub. Part No. 13778.pps

Size: 0.1 mL 0.3 mL 0.75 mL 1.5 mL 15 mL

Pub. No. MAN0001182

Store at 4°C (do not freeze)

Rev. Date 20 July 2012

Description

· Lipofectamine® RNAiMAX Transfection Reagent is a proprietary formulation for transfecting small RNAs (e.g., siRNA, Silencer® Select siRNA, Stealth® RNAi, mirVanaTM miRNA mimics and inhibitors) into a wide range of eukaryotic cells. Nucleic acid-Lipofectamine® RNAiMAX complexes can be added directly to cells in culture medium, in the presence or absence of serum. It is not necessary to remove complexes or change/add medium after transfection. Use Opti-MEM® I Reduced Serum Medium (Cat. no. 31985-062) to dilute Lipofectamine® RNAiMAX Transfection Reagent and RNA. Resuspend siRNA with nuclease-free water at a recommended stock concentration of 100 M. Store aliquots at ­20°C. Dilute stock siRNA to a working concentration of 10 M for transfection. A 10 M stock of siRNA duplex is equivalent to 10 pmol/L. Use 30 nM RNAi duplex as a starting point. BLOCK-iTTM Alexa Fluor® Red Fluorescent Oligo (Cat. no. 14750100) can be used to determine transfection efficiency. For additional information and protocols, refer to the manual at www.lifetechnologies.com/transfection.

· ·

Important Guidelines for Transfection

· · · ·

·

For research use only. Not for human or animal therapeutic or diagnostic use.

Cells

Seed cells so they will be 60­80% confluent at the time of transfection.

Day 0

Lipofectamine® RNAiMAX Reagent

Dilute Lipofectamine® RNAiMAX Reagent in Opti-MEM® Medium.

siRNA

Dilute siRNA in Opti-MEM® Medium.

Day 1

Add diluted siRNA to diluted Lipofectamine® RNAiMax Reagent (1:1 ratio).

5

Incubate.

Add siRNA-reagent complex to cells.

Day 2­4

Visualize/analyze transfected cells.

RNAi Transfection

Transfect cells according to the following table. The transfection is designed for 1 RNA amount combined with 1 amount of Lipofectamine® RNAiMAX. The prepared mix is enough to have triplicates (96-well), duplicates (24-well), and single well (6-well) transfections, and account for pipetting variations. Component

Adherent cells Opti-MEM® Medium Lipofectamine RNAiMAX Reagent Opti-MEM Medium siRNA (10 µM) Diluted siRNA Diluted Lipofectamine® RNAiMAX Reagent

® ®

96-well

1­4 x 104 25 µL 1.5 µL 25 µL 1.5 µL (15 pmol) 25 µL 25 µL

24-well

0.5­2 x 105 50 µL 3 µL 50 µL 3 µL (30 pmol) 50 µL 50 µL

6-well

0.25­1 x 106 150 µL 9 µL 150 µL 9 µL (90 pmol) 150 µL 150 µL

Incubate for 5 minutes at room temperature siRNA-reagent complex/well 10 µL 50 µL 250 µL

Incubate cells for 1­3 days at 37°C

The following table shows the amount of siRNA and Lipofectamine® RNAiMAX Reagent per well in each transfection reaction. For additional information on scaling your transfection reaction, see page 4. Amount

siRNA/well Lipofectamine RNAiMAX Reagent/ well

®

96-well

3 pmol 0.3 µL

24-well

15 pmol 1.5 µL

6-well

75 pmol 7.5 µL

Scaling Up or Down Transfections

Use the following table to scale the volumes for your transfection experiment.

Culture Vessel 96-well 48-well 24-well 12-well 6-well 60-mm 10-cm T75 T175 Growth area/ well 0.32 cm2 0.95 cm2 1.9 cm2 3.8 cm

2

Multiplication factor1 0.17 0.50 1.00 2.00 5.00 11.05 28.95 39.47 92.11

Vol. plating growth medium 100 µL 250 µL 500 µL 1 mL 2 mL 5 mL 10 mL 15 mL 35 mL

Vol. complex Opti-MEM per well 2 × 5 µL 2 × 12.5 µL 2 × 25 µL 2 × 50 µL 2 × 100 µL 2 × 250 µL 2 × 500 µL 2 × 750 µL 2 × 1.75 mL

RNA

Lipid reagent2 0.3 µL 0.75 µL 1.5 µL 3 µL 7.5 µL 17 µL 43 µL 59 µL 138 µL

3 pmol 7.5 pmol 15 pmol 30 pmol 75 pmol 166 pmol 434 pmol 592 pmol 1382 pmol

9.5 cm2 21 cm2 55 cm2 75 cm

2

175 cm2

1 After determining the optimum reagent amount, use the multiplication factor to determine the reagent amount needed for your new plate format. 2 Optimum amount needed is determined from the protocol (see pages 2­3).

Reverse Transfection of RNAi

Reverse transfection is faster to perform than forward transfection and is the method of choice for high-throughput transfection. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. Because the cells and siRNA-reagent complexes are prepared on the same day, it is recommended to use 2.5X more cells than for a regular transfection.

Limited Product Warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at www.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support.

Disclaimer: LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR

IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

©2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

For support visit www.lifetechnologies.com/support www.lifetechnologies.com

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