Introduction

The BLOCK-iT™ Fluorescent Oligo is a fluorescein-labeled dsRNA oligomer designed for use in RNAi analysis to facilitate assessment and optimization of cationic lipid-mediated delivery or electroporation of dsRNA oligonucleotides into mammalian cells.  Using the BLOCK-iT™ Fluorescent Oligo in RNAi studies offers the following advantages:

  • Provides a good indication of the transfection efficiency with Invitrogen’s Stealth™ RNAi, standard unmodified siRNA, or purified Dicer-generated siRNA.
  • Allows strong, easy fluorescence-based indication of transfection efficiency in every RNAi experiment.


Characteristics of the BLOCK-iT™ Fluorescent Oligo

The BLOCK-iT™ Fluorescent Oligo possesses the following characteristics:

  • Is a fluorescein-labeled, double-stranded RNA duplex with the same length, charge, and configuration as standard siRNA.
  • Contains chemical modifications that enhance the stability and allow assessment of fluorescence signal for a significantly longer time period than is obtained with other unmodified, fluorescently labeled RNA.

    Example:  Fluorescence signal is readily detectable in HEK293 cells for at least 72 hours.  Note that the strength of the fluorescence signal depends on the transfection efficiency and growth rate of the cells.
  • The sequence of the BLOCK-iT™ Fluorescent Oligo is not homologous to any known gene, ensuring against induction of non-specific cellular events caused by introduction of the Oligo into cells.
  • Localizes primarily to the nucleus upon uptake(1). 
  


Important:   The BLOCK-iT™ Fluorescent Oligo is designed strictly for use as a tool for siRNA uptake assessment, and is not meant to provide any information about the behavior of your Stealth™ RNAi or siRNA including its cellular localization, half-life, or stability.

Materials

The BLOCK-iT™ Fluorescent Oligo is supplied as a 20 µM or 1 mM stock of fluorescein-labeled double-stranded RNA (dsRNA) oligomer in 100 mM KOAc, 30 mM HEPES-KOH, pH 7.4, and 2 mM MgOAc.

Protocol

Handling the BLOCK-iT™ Fluorescent Oligo

The BLOCK-iT™ Fluorescent Oligo is supplied as a 20 µM or 1 mM stock solution in an annealing buffer.  Follow the guidelines below when handling the BLOCK-iT™ Fluorescent Oligo stock solution.
 
  1. The BLOCK-iT™ Fluorescent Oligo is light sensitive.  Store the stock solution at -20°C, protected from light. The stock solution is stable for at least 6 months if stored properly.

  2.  When using, thaw the stock solution on ice or at room temperature.  Once thawed, place the tube on ice until use.  After use, return stock solution to -20°US C storage.

  3. The stock solution may be frozen and thawed multiple times without loss of fluorescence signal if handled properly.

  4. Take precautions to ensure that the stock solution does not become contaminated with RNase

  • Use RNase-free sterile pipette tips and supplies for all manipulations.
  • Wear gloves when handling reagents and solutions.

Using the BLOCK-iT™ Fluorescent Oligo for Cationic Lipid-Mediated Transfection

You may use the BLOCK-iT™ Fluorescent Oligo (20 µM or 1 mM stock) with any cationic lipid-based transfection reagent suitable for delivery of Stealth™ RNAi, siRNA, or Dicer-generated siRNA to mammalian cells.  Follow the guidelines below when transfecting the BLOCK-iT™ Fluorescent Oligo.
 
Tip:   For highly efficient transfection of a wide variety of mammalian cells, we recommend using Lipofectamine™ 2000 Reagent (Catalog no. 11668-027) available from Invitrogen(2).

  • The amount of BLOCK-iT™ Fluorescent Oligo to use depends on the growth rate and transfection efficiency of the mammalian cells.  If you are transfecting your mammalian cell line for the first time, we recommend evaluating several concentrations of lipid and varying the final concentration of the BLOCK-iT™ Fluorescent Oligo from 10 to 200 nM to determine the optimal amount of BLOCK-iT™ Fluorescent Oligo to use to obtain a strong fluorescence signal.

    Note:  For most cell lines tested (e.g. HEK293, A549, HeLa), we obtain a readily detectable fluorescence signal when using 100 nM BLOCK-iT™ Fluorescent Oligo for transfection.

  • Prepare and seed mammalian cells at a density recommended by the manufacturer of the transfection reagent you are using.
  • Prepare lipid-BLOCK-iT™ Fluorescent Oligo complexes as directed by the manufacturer of the transfection reagent you are using.  Always dilute the BLOCK-iT™ Fluorescent Oligo immediately before transfection (i.e. do not store diluted Oligo) and into an appropriate medium.  We recommend diluting the BLOCK-iT™ Fluorescent Oligo into Opti-MEM I Reduced Serum Medium (Catalog no. 31985-062) available from Invitrogen.
  • Assess fluorescent uptake at 6 to 24 hours post-transfection.  The fluorescence signal may be detected at longer time points depending on the transfection efficiency and growth rate of the cells.

Using the BLOCK-iT™ Fluorescent Oligo for Electroporation

When performing electroporation, higher concentrations of BLOCK-iT™ Fluorescent Oligo may be required. Use the 1 mM stock solution of BLOCK-iT™ Fluorescent Oligo to optimize electroporation conditions according to the manufacturer’s guidelines.

Detecting Fluorescence Signal

Once you have transfected or electroporated your mammalian cells with the BLOCK-iT™ Fluorescent Oligo, you may qualitatively assess Oligo uptake in live cells using fluorescence microscopy.  You may use any type of fluorescence microscope and a standard FITC filter set (lambdaex = 494 nm, lambdaem = 519 nm green) for detection.

References

  1. Fisher, T.L., Terhorst, T., Cao, X., and Wagner, R.W. (1993) Nuc. Acids Res. 21, 3857-3865.
     
  2. Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P., Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S. (1999) Focus 21, 54-55.  ©2005 Invitrogen Corporation. All rights reserved.
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