RNA Detection

denaturation at 98C for 10 s, annealing at 55C for 30 s, and

extension at 68C for 30 s.

3. Precipitate the DNA by adding a one-tenth volume of 3 M
   sodium acetate and a 1.1 volume of isopropanol. Centrifuge
   the sample at 15,000gfor 10 min to pellet the DNA.


4. Remove the supernatant carefully, and rinse the pellet with 70%
   ethanol.


5. Air-dry the pellet for 5 min, and dissolve it in a 20-μL TE buffer
   solution.

3.3.2 Preparation of RNA
Pools

1. Prepare the in vitro transcription reaction at room temperature
   in the order as described in the Materials.


2. Incubate the reaction mixture overnight at 37C.


3. Add 1μL of DNase and incubate for 15 min at 37C.


4. Precipitate the RNA by adding a one-third volume of 10 M
   ammonium acetate and a 1.5 volume of isopropanol. Centri-
   fuge the sample at 15,000gfor 10 min at 4C to pellet the
   RNA.


5. Thoroughly remove the supernatant, and dissolve the pellet in
   0.5 mL of RNase-free water.


6. Apply the sample to the preequilibrated NAP-5 column,
   and elute the sample with 1 mL of RNase-free water
   (seeNote 10).


7. Precipitate the RNA by adding a one-tenth volume of 3 M
   sodium acetate and a 1.1 volume of isopropanol. Centrifuge
   the sample at 15,000gfor 10 min to pellet the RNA.


8. Remove the supernatant, and rinse the pellet with 70% ethanol.


9. Air-dry the pellet for 5 min, and dissolve it in 100μL of the
   annealing buffer.


10. Use the solution as an RNA pool for in vitro selection.

3.3.3 In Vitro Selection of
RNA Aptamer to BHQ1-
Immobilized Resin (See
Note 11)

1. Place 20μL of the BHQ1-immobilized resin (50% slurry) in an
   empty centrifugal filter unit, and wash the resin three times
   with 400μL of the annealing buffer.


2. Apply the RNA pool solution to the resin, and incubate on ice
   for 30 min with manual shaking every 3 min.


3. Drain and wash the resin with annealing buffer to remove
   unbound RNAs.


4. Elute the bound RNAs 3 times with 130μL of the annealing
   buffer saturated with free BHQ1 amine.


5. Precipitate the RNAs by adding a one-tenth volume of 3 M
   sodium acetate and a 1.1 volume of isopropanol. Centrifuge
   the sample at 15,000gfor 10 min to pellet the RNA.

312 Shin-ichi Sato et al.