RNA Detection

3 Methods

3.1 Preparation of
Reference Dye-
Labeled Controls (See
Note 5)

1. React 150μL of the aminodextran solution with IRDye® 800
   NHS ester at a dye to dextran molar ratio of 3:1 on a thermo-
   mixer overnight at 25C.


2. Purify the mixture on a NAP-5 gel chromatography column in
   elution buffer to remove unbound dye.


3. Concentrate the purified IRDye®800-labeled dextran solution
   on a centrifugal filter unit.


4. Determine the concentration of IRDye® 800 fluorophore
   using a spectrophotometer.

3.2 Establishment of
In Vitro Standard
Measurements

To create aqueous bubbles containing the IRDye®800-labeled

dextran and nonhybridized or fully hybridized MBs, water-in-oil

emulsions are prepared as follows:

1. On an electronic scale, tare a 50 mL tube. Add 24 g of mineral
   oil.


2. Use a glass pipette to add 447 mg of Span 80 dropwise to the
   mineral oil.


3. Vortex the mixture vigorously.


4. Use a glass pipette to add 54 mg of Tween 80 dropwise to the
   mineral oil.


5. Vortex the mixture vigorously.


6. Add a 3 mL aliquot of the above mixture to a glass vial and stir
   using a magnetic stirrer. Make sure the stir bar is immersed
   under the emulsion surface. Stir for 1–2 min (seeNote 6).


7. While stirring, add 1–2μL of aqueous samples containing 5μM
   nonhybridized or fully hybridized MBs (previously hybridized
   with 20μM RNA target for at least 1 h at room temperature)
   and 1μM IRDye®800-labeled dextran in a dropwise fashion
   into the emulsion to form water-in-oil microemulsion bubbles.


8. After stirring for 3–5 min more, take an aliquot of the emulsion
   sample and place it on a glass bottom dish for microscopy
   imaging and analysis (seeNote 7).


9. Example images of microemulsion bubbles acquired using the
   Cy5 and IRDye®800 filter sets are shown in Fig.1 (seeNotes
   8 and 9 ).

Microporation is used to deliver MB–dextran mixtures because it

enables rapid and highly efficient delivery of MBs and dye-labeled

Optimizing Molecular Beacons 247