Supporting Information
Strategies of fluorescence staining for trace total ribonucleic acid analysis by capillary
electrophoresis with argon ion laser-induced fluorescence
Yi-An Chung, Yi-Hsin Chen, Po-Ling Chang*
Department of Chemistry, Tunghai University, Taichung, 40704 Taiwan
Materials and methods
Chemicals
Poly(vinylpyrrolidone) (PVP, average Mw 1,300,000 Da), poly(ethylene oxide) (PEO,
average Mw 4,000,000 Da) and the chemicals used to prepare the buffer solution were
obtained from Sigma-Aldrich (St. Louis, MO, USA). The asymmetric cyanine dyes
(SYTO-9, SYBR Green I, SYBR Green II, SYBR Safe, SYBR Gold) and DEPC and
DNase/RNase-free water were obtained from Life Technologies (Carlsbad, CA, USA).
Cell culture, RNA extraction
The NPC cells (HK-1) were kindly provided by Prof. S.-J. Chen (Molecular Medicine
Research Center, Chang Gung University). Briefly, the cells were cultured in RPMI
medium at 37°C in 5% CO2 for three days. The total RNA was extracted from the cell
lines using TRIzol Reagent in accordance with the manufacturer’s instructions (Life
Technologies, NY, USA). The extracted RNA was dissolved in DEPC water and kept
at –80ºC for long-term storage. Before injection into the capillary, the RNA samples
were heated to 70°C for 5 min and then transferred to ice box to avoid crosshybridization between RNA samples. DNase/RNase-free water was used to adjust the
concentrations of total RNA. The RNA samples remained stable for several months at
–20°C. Prevention of repeated freeze-thaw cycles of stock RNA samples must be taken
into account due to the decrease of the RNA integrity observed when freeze-thawing
the same RNA sample more than three times.
Fluorescence measurements
The intrinsic fluorescence intensities of dyes (dissolved in 1x PCR buffer, pH 8.0) were
measured by a Cary Eclipse Fluorescence Spectrophotometer (Agilent, Foster City, CA,
USA). To compare the enhancement of DNA/dye fluorescence, the dyes were added a
divided PCR products with a length of 342 bp [1-3]. Briefly, 49 μL PCR product (in 1x
PCR buffer, pH 8.0) was mixed with 1 μL dye (SYTO-9 25 μM, 50x SYBR Green I,
50x SYBR Green II, 50x SYBR Safe, 50x SYBR Gold) and gently vortexed by a
oscillator followed by transfer to a 50 μL quartz cuvette. The fluorescence
measurements were performed by 488 nm excitation and the fluorescence spectrum up
to 800 nm was recorded at ambient temperature. The spectral areas from 495 to 650 nm
were used to calculate the intensity and enhancement of the fluorescence.
Capillary electrophoresis with laser-induced fluorescence
A CE-LIF system built in-house was used in our laboratory. Briefly, a high-voltage
power supply (Gamma High Voltage Research Inc., Ormond Beach, FL, USA) was
used to drive electrophoresis. The entire detection system was enclosed in a black box
with a high-voltage interlock. The high-voltage end of the separation system was placed
in a plastic bottle for safety. A 488 nm argon ion laser from JDS Uniphase (Milpitas,
CA, USA) with 10 mW total radiated power was used for excitation. The laser beam
was focused into the capillary by a PCX lens (Edmand, Barrington, NJ, USA). The
fluorescence was collected with a 10× objective (numerical aperture = 0.25), and an
appropriate interference filter (SYTO-9: 500 nm, SYBR Green I: 520 nm, SYBR Green
II: 505 nm, SYBR Safe: 520 nm, SYBR Gold: 535 nm) combined with a GG 495 longpass filter was arranged after the objective to reject the scattered light before the emitted
light reached the photomultiplier tube (R928, Hamamatsu Photonics, Hamamatsu,
Japan). All of the filters utilized in this work were obtained from Edmund (Barrington,
NJ, USA). The amplified currents were transferred directly through a 10 k resistor to
a 24-bit A/D interface at 10 Hz, which was controlled using chromatographic software
(Clarity, DataApex, Prague, Czech Republic) and stored in a personal computer. Bare
fused-silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) with a 75 m
internal diameter were used for separation and coated with 5% PVP overnight at
ambient temperature. The polymer for the capillary coating was dissolved in double
deionized H2O (ddH2O). The capillary length was 40 cm, and the effective length from
the detector was 33 cm. Anodic and cathodic vials were filled with a 0.4% PEO solution
that was prepared in 2x TGA buffer (14 mM Tris, 192 mM glycine, 10 mM acetic acid,
pH 7.5)[4, 5] containing SYTO-9 (0.5 μM), SYBR Green I (1x), SYBR Green II (1x),
SYBR Safe (1x) or SYBR Gold (1x) for on-column staining. In the pre-column stain
mode, the fluorescent dye was not contained in the polymer solution; each dye was
added to the RNA sample and incubated at 70°C for 5 min followed by cooling in an
ice box for 1 min. The RNA samples were then introduced into the capillary by
electrokinetic injection for 10 sec at 5-20 kV.
Table S1 Optical characteristics and performance of five fluorescent dyes for DNA and RNA staining.
SYTO 9
SYBR Green I
SYBR Green II
SYBR Safe
SYBR Gold
484/501
498/520
488/504
509/527
497/539
Quantum yieldsa
0.6
0.8
0.36/0.54
N.A.
0.7
Free dye intensitiesb
17
18
50
12
44
DNA/dye intensitiesb
4799
10369
5984
3034
6315
Enhancementsc
282
576
120
253
144
On-column stain intensitiesd
2.1
2.0
3.7
1
5.1
Pre-column stain intensitiesd
1
16.9
5.6
5.2
143.1
Wavelengths (λex/λem)
a.
b.
c.
d.
The quantum yields were obtained from reference [6] and [7] and N.A. means not available.
The intensities were calculated by integral from area of fluorescence spectra from 495 to 650 nm.
The enhancement values are the ratio of fluorescence intensity of free dye and DNA/dye complex.
The intensities are sum of peak area of 28S and 18S rRNA in the electropherograms and normalized to the smallest one (on-column stain:
SYBR Safe, pre-column stain: SYTO 9).
Figure S1. Comparison of the fluorescence spectra of a) SYBR Safe, b) SYTO-9, c)
SYBR Green II, d) SYBR Green I and e) SYBR Gold. Each dye was dissolved in 1x
PCR buffer to measure the fluorescence spectra. All of the emission spectra were
obtained by excitation under identical wavelength (488 nm).
Figure S2. Electropherograms of total RNA with on-column fluorescence staining in
the presence of a) SYTO-9 (0.5 μM), b) SYBR Safe (1x), c) SYBR Green II (1x), d)
SYBR Green I (1x) and e) SYBR Gold (1x). HK-1 RNA (1 ng/μL) was introduced into
the capillary by electrokinetic injection at -10 kV for 10 sec. The electrophoretic
separation of total RNA was accomplished by a polymer solution (0.4% PEO, 0.1%
PVP) with separation voltage at -10 kV.
a)
Relative Fluorescence Intensity
b)
28S
c)
18S
Small RNA
tRNA
d)
e)
4
9
Migration Time (min)
14
Figure S3. The electropherograms of total RNA with on-column staining by SYBR
Gold. The concentrations of total RNA were a) 1 ng/μL, b) 0.8 ng/μL, c) 0.6 ng/μL, d)
0.4 ng/μL, e) 0.2 ng/μL. The electrophoretic conditions are the same as in Fig. S2.
a)
Relative Fluorescence Intensity
b)
c)
d)
e)
4
9
Migration Time (min)
14
Figure S4. Electropherograms of total RNA with pre-column fluorescence staining
with a) SYTO-9 (0.5 μM), b) SYBR Safe (1x), c) SYBR Green II (1x), d) SYBR
Green I (1x) and e) SYBR Gold (1x). Electrophoretic conditions were identical to
those in Fig. S2.
a)
Relative Fluorescence Intensity
b)
c)
d)
e)
4
9
Migration Time (min)
14
Figure S5. The electropherograms of total RNA with pre-column staining with SYBR
Gold (solid line) and SYTO-9 (dash line). The total RNA samples (1 ng/μL) were
introduced into capillary by electrokinetic injection for 3 second at a) -5 kV, b) -10 kV,
c) -15 kV and d) -20 kV. The electrophoretic conditions are the same as in Fig. 1.
a)
Relative Fluorescence Intensity
b)
c)
d)
4
9
Migration Time (min)
14
References
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