RNA Detection

2.2 In Vitro
Measurement
of Fluorescence
Activation Kinetics

1. Stopped-flow spectrafluorometer (e.g., Applied Photophysics
   Model SX20).


2. Spectrofluorophotometer (e.g., Shimazu RF-5300PC).


3. 50 nM and 0.2μM ECHO-liveFISH probes in PBS.


4. 0.2, 0.35, 0.7, 1.4, and 2.8μM RNA/DNA single-stranded
   oligonucleotides of targeted sequences in PBS.


5. Dilution buffer in fluorophotospectral measurement: 4SSC,
   0.5 mM EDTA, 10% dextran sulfate, and 10% deionized form-
   amide in dH 2 O.

2.3 In Vivo
Electroporation

1. Stereomicroscope.


2. Micromanipulator (e.g., Narishige MM-3).


3. Pulse generator (e.g., Nepagene NEPA21).


4. Tweezer-type cathode electrodes (e.g., Nepagene CUY650P3).


5. Adaptor cable (e.g., Nepagene C118).


6. Hook-type anode electrode (e.g., Nepagene C117).


7. Foot switch (e.g., Nepagene C200).


8. Injection microsyringe (e.g., ITO Corporation MS-NE05 with
   33-G needle connected to the anode).


9. Tweezers.


10. Gauge 27–33 needles.


11. 70% ethanol.


12. Sterile ddH 2 O.


13. 200 ng/μL ECHO-liveFISH probes in TE with 0.1% fast
    green dye.


14. 3μg/μL DsRed2-B23 expressing DNA plasmids in TE with
    0.1% fast green dye.


15. 200 ng/μL Cy5-d(T) 30 in TE with 0.1% fast green dye.

2.4 Generating Brain
Slices and Stabilizing
for Fluorescence
Imaging

1. CO 2 incubator.


2. Vibratome.


3. Stereomicroscope mounted with digital camera.


4. Confocal laser scanning microscope mounted with live-cell
   imaging chamber (e.g., Olympus FV1000).


5. Temperature and CO 2 control modules.
   6.φ27 mm-hole glass-bottom dish.


6. Slice anchor.


7. Artificial cerebrospinal fluid (ACSF):124 mM NaCl, 3 mM
   KCl, 26 mM NaHCO 3 , 2 mM CaCl 2 /2H 2 O, 1 mM
   MgSO 4 /7H 2 O, 1.25 mM KH 2 PO 4 , and 10 mMD-glucose,
   bubbled with a gas mixture of 95% O 2 /5% CO 2 before use.

262 Dan Ohtan Wang