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