RNA Detection

3.5 Fluorescence In
Situ Hybridization
(FISH) in Fixed Cells
with the RT-Aptamer
[25]

1. Fix HeLa cells on 96-well plates (3.0 103 cells per well) with
   a 4% paraformaldehyde at room temperature for 15 min.


2. Wash the cells twice with PBS.


3. Permeabilize the cells with 70% ethanol overnight at 4C.


4. Soak the cells in 2SSC buffer at 50C for 15 min.


5. Incubate the cells at 37C for 24 h with the prehybridization
   buffer.


6. Incubate the cells with 100 ng of the RT-aptamer in 2SSC
   buffer (50μL) for 2 h at 37C.


7. Treat the cells with 30μLof1μg/mL Hoechst 33342 in
   2 SSC buffer at 37C for 30 min.


8. Add a 30μLof5μM BHQ1-Cy3 probe in 2SSC buffer,
   incubate at 37C for 30 min, and then wash out excess probe
   with 2SSC buffer.


9. Observe the target RNAs under a confocal microscope (see
   Note 16).

3.6 Preparation of an
Expression Vector
Encoding RT-Aptamer
[26–28]

1. Prepare the dsDNA template encoding RT-aptamer by anneal-
   ing two 100μM synthetic ssDNA oligos, RT aptamer tem-
   plates 1 and 2 [Fig.3] in annealing buffer.


2. Prepare the ligation mixture usingBglII–HindIII linearized
   pSuper.neo plasmid as a vector, and incubate the mixture for
   30 min at 16C to allow the ligation reaction.


3. Transform DH5αwith the ligation product, and heat shock at
   42 C for 1 min and spread the cells on an LB plate containing
   100 μg/mL of carbenicillin.

GG GCU

G

G

G

CC

C

U

U

UU

A

A

A

A

A

A

U

A

U

U

A

U

G

C

C

GA

C

A

U

A

G

G

A

G

RNA Targeting Arm

Removal of

stem region

Addition of

RT arm

Recognition of

target RNA

U U

5’

3’

5 ’ 3 ’

AAUGAAGAUCAAGAUCAUUGCUCC

Target RNA

5’3’

C

U

A

C

G

A

G
G

U

A U

G

C

AAA

U

A

GC

AU

GC

U

U

U

G

CG

CG

CG

GG

G

U

• 
  

.

.

• 
  

5’ 3’

AUGCAGGA AG

C

UA

C

G

A

G

G

U

A

U

G

C

AAA

U

UU U

CG

G

UAUACUUCUG

AAUGAAGAUCA UAC UUGCUCC

AGA

3’. 5’

• 
  


• 
  

  ---

  
  


• 
  


• 
  


• 
  


• 
  


• 
  


• 
  

GG GCU

G

G

G

CC

C

U

U

UU

A

A

AA

A

A

U

Fig. 2Preparation of the BHQ1-binding RNA aptamer

314 Shin-ichi Sato et al.