Analytical Chemistry Chemical Cytometry Quantitates Superoxide

quantum efficiency of fluorescence dyes may be optimized as
reported by Nie and co-workers. 56
Quantification of miR-21 in Normal and Cancerous Cell
Lines. MCF-7 (Invasive breast ductal carcinoma cell line) and
HepG2 (human hepatocarcinoma cell line) are regarded as a
common breast and liver cancer cell line model, respectively, while
HUVEC (human umbilical vein endothelial cells) is regarded as
normal cell line. Herein, we employed the single-molecule detection
assay to quantify differential-expressing miRNAs in normal
and cancerous cell lines. Briefly, total RNA of each cell line was
extracted prior to the direct miRNA detection. The YOYO dye
has no selectivity toward oligonucleotides, mRNAs, tRNAs, and
other small RNAs in such environment. To eliminate the signals
drawn from the complex matrixes without supplementary pretreatments
on the cell samples, standard addition method is
adopted in the manner that synthetic target miR-21 is spiked to
the mixture of total RNA sample and probes. The original
concentration of miR-21 in the sample of total RNA of each cell
lines can eventually be obtained by extrapolation of the calibration
curve. Three independent standardization curves were prepared
for the three cell lines and the amounts of miR-21 in each of the
cell line were determined. All three calibration curves showed
strong correlation between the numbers of counted molecules and
the miRNA concentration with the coefficients of determination
for all three of them greater than 0.997 (Figure 5A-C). Since the
hybridization efficiency was ∼80% as mentioned previously, the
concentration of miR-21 in each cell line estimated by standard
addition method was multiplied by the efficiency conversion factor
of 1.25 such that the actual miR-21 contents in total RNA can be
obtained. The contents of miR-21 in total RNA of each cell line
were found to be 0.92, 0.43, and 0.32 amol/ng for MCF-7, HepG2,
and HUVEC, respectively.
For the purpose of result validation, we quantified the content
of miR-21 in the three cell lines using the same batch of cells by
qRT-PCR method. Quantitative RT-PCR is a technique commonly
adopted as a standard method for miRNA profiling. It is superior
to other detection assays for its high specificity and minute
amounts of starting materials used in the detection. Amplification
steps however are involved and it takes approximately 5 h for the
whole process. The output of qRT-PCR is usually expressed in
terms of fold-change and so the raw data is semiquantitative.
Additional calibration curve has to be established for quantitation
purpose. To compare, our SMD detection assay is relatively rapid
as it takes only 1hofsample incubation and promptly followed
by microscopic detection. The result is quantitative and obtained
by applying standard addition methods. The standardization curve
(56) Nie, S. M.; Chiu, D. T.; Zare, R. N. Anal. Chem. 1995, 67, 2849–2857.
6918 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
by qRT-PCR is shown in the Supporting Information (Figure S5).
Figure 5D displays the contents of miR-21 in MCF-7, HepG2, and
HUVEC cells determined by the SMD assays and those by qRT-
PCR with calibration standardization. The SMD result agrees very
well with the outcome of qRT-PCR. The high correlation with the
accredited qRT-PCR methods demonstrated that the pretreatmentfree
SMD system developed here is of high potential in profiling
expression of miRNAs in different cell lines and thus applicable
in early cancer diagnosis.
CONCLUSIONS
We developed a direct and amplification-free quantitative assay
of single miRNA molecules using solution-based hybridization
approach and fluorescence-based detection with TIRFM. This
assay is straightforward, rapid, and highly sensitive. The fluorescent
hybrids diffuse randomly in the refined detection volume.
Improvement on the limit of detection could be achieved by
increasing sampling time or immobilizing target fluorescent
hybrids onto the coverslips. As a proof of concept, the content of
miR-21 were determined in cancerous MCF-7 and HepG2 and
noncancerous HUVEC cell lines and the result agreed very well
with that of conventional qRT-PCR. Both the success in discriminating
differentially expressing miR-21 in different cell lines and
the high correlation with the conventional detection method
justified the potential of the system in application for future early
cancer diagnosis.
ACKNOWLEDGMENT
This work was fully supported by the Faculty Research Grant
of Hong Kong Baptist University (FRG/07-08/II-68) and grant
from the University Grants Council of the Hong Kong Special
Administrative Region, China (HKBU I/06C). We thank Dr.
C. K. C. Wong from the Department of Biology of HKBU for
providing the MCF-7 cells.
SUPPORTING INFORMATION AVAILABLE
Additional information includes video of miRNA diffusing in
probe volume, adsorption time analysis of single hybrid molecules,
pixel size analysis of hybrids prepared in TE buffer containing
250 and 500 mM NaCl, MALDI-TOF mass spectrum of LNA
strands, calibration curve of qRT-PCR. This material is available
free of charge via the Internet at http://pubs.acs.org.
Received for review April 30, 2010. Accepted July 14,
2010.
AC101133X