RNA Detection

PUM-HD mutant; PUM-HD was mutated to address the

sequence UUAGGGUU (designated as mPUMt). Telomeres

were visualized using a fusion protein of iRFP and TRF1. Then

hnRNPA1 was fused to a SNAP tag and was conjugated with a red

fluorescent dye, tetramethylrhodamine (TMR). We introduced the

genes of these three probes into living cells and observed them

using TIRF microscopy. Herein, we describe details of methods for

this sample preparation and observation.

2 Materials

2.1 Plasmids (See
Note 1)

1. NLS-GN-mPUMt-GC/pcDNA3.1(+).


2. hnRNPA1-SNAPf/pcDNA3.1(+).


3. iRFP-TRF1/pCLNCX.


4. pCL-10A1 (a packaging vector in RetroMax system).

2.2 Cell Culture and
Transfection

1. Gag293 cells.


2. U2OS cells.


3. Dulbecco’s modified Eagle’s medium (DMEM) supplemented
   with 10% fetal bovine serum (FBS).


4. Trypsin solution (0.005 w/v %).


5. Opti-MEM.


6. Lipofectamine 2000 (Invitrogen Corp.).


7. Lipofectamine LTX (Invitrogen Corp.).


8. Polybrene.


9. Phosphate-buffered saline (PBS). Autoclaved before storage at
   room temperature.


10. Glass-bottomed dish (e.g., Asahi Glass Co., Japan).


11. CO 2 incubator.

2.3 Fluorescence
Microscopy
Observation

1. Hanks’ balanced salt solution (HBSS).


2. MEM nonessential amino acids solution (100, Gibco).


3. Imaging medium: HBSS containing 1MEM nonessential
   amino acids solution, 2.5 g/L glucose, 2 mM glutamine,
   1 mM sodium pyruvate, and 10 mM HEPES pH 7.4.


4. 1μM SNAP-TMR.


5. Inverted fluorescence microscope, (e.g., Olympus IX81),
   equipped with a 1001.49 NA oil immersion objective, and
   a home-built excitation optical system.


6. EM-CCD camera (e.g., ImagEM; Hamamatsu Photonics
   K.K.).

Spatiotemporal Imaging of Single Telomeric-Repeat Containing RNA 339