Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 1. Schematic illustration of the hybridization-based TIRFM assay for the detection of single miRNA molecules in solution.
Bio-Rad iCycler, Version 4.006 (Bio-Rad Laboratories, Inc., Hercules,
CA). The PCR reactions were run along with no-template
control and RT-minus control. Data were analyzed by iCycler iQ
Optical System Software, Version 3.0a (Bio-Rad Laboratories), and
the miRNA expression level was measured using threshold cycle
(Ct) which is the cycle number at which the fluorescence
generated within a reaction crosses the threshold. The Ct values
were then converted to absolute amount using a standard curve
of mature miR-21. Six independent experiments were performed,
and each experiment was run in duplicate.
Establishment of miR-21 Calibration by qRT-PCR. A
mixture of synthetic RNA oligonucleotides from mirVana miRNA
Reference Panel v9.1 (Applied Biosystems) was used to generate
a standard curve for miR-21. The RNA oligonucleotides representing
mature miR-21 ranging from 10 -6 to 1 fmol were reverse
transcribed, also using TaqMan MicroRNA Reverse Transcription
Kit (Applied Biosystems) and mature miR-21-specific
TaqMan MicroRNA Assays (P/N: 4373090, Applied Biosystems).
The PCR amplification of the cDNA was then performed
using same materials as mentioned above. Three independent
experiments were performed, and each experiment was run
in duplicate. Calibration curve was shown in Supporting
Information.
Quantification of miR-21 in Cells by TIRFM. Total RNA
of HUVEC, HepG2, and MCF-7 was diluted to 150 ng/µL with
TNE buffer, respectively. Consequently, 7.5 µL of diluted total
RNAs were spiked into a mixture standard solution of miR-21 of
a final concentration of 0, 1, 2, 5, 10, and 15 pM and a LNA probe
with a final concentration of 100 pM, and finally diluted with TNE
buffer to a volume of 44 µL. The solution was incubated for 1hat
52 °C and followed by addition of 1 µL of YOYO dye as mentioned
previously. After equilibrium for 5 min, 10 µL (with 250 ng of total
RNA) of solution was pipetted to coverslips for TIRF imaging and
quantitation. The contents of miR-21 in the three cell lines were
estimated by standard addition method and the value of miR-21
quantified by the TIRFM platform was compared with the outcome
of the qRT-PCR method.
Imaging System. An inverted Olympus IX-71 microscope
(Olympus, Tokyo, Japan) was equipped with a high-numerical
aperture 60× oil-immersion objective (1.45 NA, PlanApo, Olympus)
as shown in Figure S1A. The sample coverslip was located under
the fused-silica Isosceles Brewster Prism (CVI Melles Griot,
Carksvadm, CA) and above the 60× objectives with immersion
oil (η ) 1.52, Nonfluorescence, Olympus) in between. A 488 nm
cyan laser (50 mW, CMA1-01983, Newport, NJ) was used as the
excitation source. The laser beam was first filtered with neutral
6914 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
density filter (FSR-OD 60, Newport), focused by cylindrical lens
(focal length, 150 mm; CVI Melles Griot), and eliminated with
the aid of pinholes before its entry to the prism with an incident
angle of approximately 66°. Evanescent field was generated by
the TIR of laser beam occurring at prism and was used to excite
the fluorescently labeled hybrid molecules. A band-pass emission
filter (D535/40 m Chroma Technology) was placed between the
objective lens and the EMCCD to collect emitted photons. A
uniphase mechanical shutter (model LS272, Vincent Associates,
Rochester, NY) and a driver (model VMM-T1, Vincent Associates)
were synchronized with the PhotonMax: 512B electron-multiplied
CCD camera (EMCCD, Princeton Instruments, Princeton, NJ) in
external synchronization mode and frame-transfer mode. The
mechanical shutter blocked laser beam when the camera was off
in order to reduce photobleaching. The ADC rate of the camera
was 10 Hz, exposure time was 50 ms, and the multiplication gain
was set at 4000, with the shutter driver set to 100 ms exposure
and 100 ms delay. Typically, an image series of 10 sequential
frames on 10 locations were acquired from a single slide. Images
were obtained with the WinSpec/32 software (Version 2.5.22.0,
Downingtown, PA) provided by Princeton Instruments.
Data Analysis. All captured images were analyzed with a
public-domain image-processing program Image J (version 1.43i,
NIH, Bethesda, MD). A region of interest (ROI) with 200 pixelssquare
at the center of the light spot with relatively even laser
intensity was selected for single molecule counting. Intensity of
images may also suffer while the shutter was triggered on or off.
Five subframes (frame 3 to frame 7) of the image series were
selected for analysis. The threshold for image acquirement was
chosen at a value of three times the standard deviation of the mean
intensity of the image. The image was then further processed with
the Analyze Particles function in Image J to determine the number
of single fluorescence particles computationally. The size of
particles was set at 2-10 pixels to reduce false positive signals
generated from noises. Number of spots in five frames was
counted separately and summed up (i.e., accumulation of spots
imaged within 250 ms). Consequently, the sum of spots from 10
image series of a single slide was averaged. All experiments were
done in triplicate and the error bars of charts shown in the Results
and Discussion refer to the standard error of mean of the triplicate
experiments unless specified.
RESULTS AND DISCUSSION
In this study, the miRNA detection is based on the hybridization
approach as illustrated in Figure 1. Complementary probes
of oligonucleotides were hybridized with the target miR-21 in