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 Pdf
file on Development of a low-cost detection method for miRNA
microarray
Wei Li, Botao
Zhao, Youxin Jin, and Kangcheng
Ruan*
State Key
Laboratory of Molecular Biology, *Correspondence
address. Tel: +86-21-54921168; E-mail: [email protected]
MicroRNA (miRNA) microarray is a
powerful tool to explore the expression profiling of miRNA.
The current detection method used in miRNA
microarray is mainly fluorescence based, which usually requires costly
detection system such as laser confocal scanner of
tens of thousands of dollars. Recently, we developed a low-cost yet sensitive
detection method for miRNA microarray based on
enzyme-linked assay. In this approach, the biotinylated miRNAs
were captured by the corresponding oligonucleotide
probes immobilized on microarray slide; and then the biotinylated miRNAs
would capture streptavidin-conjugated alkaline phosphatase.
A purple-black precipitation on each biotinylated miRNA
spot was produced by the enzyme catalytic reaction. It could be easily
detected by a charge-coupled device digital camera mounted on a microscope,
which lowers the detection cost more than 100 fold compared with that of
fluorescence method. Our data showed that signal intensity of the spot
correlates well with the biotinylated miRNA
concentration and the detection limit for miRNAs
is at least 0.4 fmol and the detection dynamic
range spans about 2.5 orders of magnitude, which is comparable to that of
fluorescence method.
Keywords
microRNA; miRNA microarray;
enzyme-linked detection method of miRNA
Received:
December 30, 2009 Accepted: January 13, 2010
Introduction
MicroRNAs (miRNAs)
are single-strand non-coding RNAs of about 22
nucleotides in length. They are generated from hairpin structure precursors (
pre–miRNA) through the cleavage of Dicer
enzyme [1,2]. miRNAs
exist widely in animals and plants and play important regulatory roles through
targeting mRNAs for cleavage or translational repression [1,3]. They are known to participate
in many biological processes, including development, differentiation,
apoptosis, etc. [4–7]. Many miRNAs have been shown to be associated
with human diseases, such as breast cancer [8], cardiac
disease [9], leukemia [10], etc. All these studies
indicated that miRNA expression levels are closely
associated with developmental stages and physiological states as well as
disease processes and thus miRNA expression
profiling plays an important role in miRNA
studies. And among all the miRNA expression
profiling approaches, the microarray method has been widely used because of
its high throughput, small sample volume requirement, etc. [11–19]. In the current field of miRNA microarray,
fluorescence detection method has been mainly used, in which a costly
detection instrument such as laser confocal
microarray scanner is required [11,18,19]. The high cost will limit the wide applications of miRNAs
microarray, especially in clinical diagnosis. Recently, we established a
low-cost detection method for miRNA microarray
using an enzyme-linked assay that is similar to Joos’ ELISA detection method used in protein microarrays
[20]. In our method, alkaline phosphatase
was introduced through its streptavidin conjugate
binding to biotinylated miRNA, which was
specifically captured by its corresponding oligonucleotide
probes immobilized in a spot on microarray slides. The purple-black
precipitation of the enzyme catalytic product on the spot could be easily
detected by a charge-coupled device (CCD) digital camera mounted on a
microscope and the detection limit for miRNAs was
at least 0.4 fmol and the detection dynamic range
was from 70 to 35 nM spanned about 2–3 orders of magnitude. Using this method, we analyzed the
expression of eight miRNAs in epididymis
of 9-week-old rat and found that this method had good reproducibility and the
detection results were consistent with that of the fluorescence detection
method.
Materials and Methods
Materials
Standard
microscope glass slides (
Principle
of the enzyme-linked detection for miRNA
microarray
Figure
1 shows the schematic diagram of enzyme-linked detection
method of miRNA microarray. The isolated miRNAs
from biological samples were first biotinylated at the 3‘ terminal and then were captured, respectively, by the corresponding miRNA
probes in hybridization process. The miRNA probes
were pre-spotted on the slides to form a miRNA
microarray. After the removal of the unbound miRNAs,
streptavidin-conjugated alkaline phosphatase
was added to react with the biotinylated miRNAs
captured at each spot on the microarray slides. Finally, the microarray is
incubated with BCIP/NBT, in which BCIP can be hydrolyzed by the catalysis of
alkaline phosphatase to produce an intermediate
that is then oxidized by NBT to form insoluble dark-purple materials
precipitated on the spots of the slides. In this process, the enzyme as an
amplifier converts the trace miRNA into a large
number of precipitations. Alkaline phosphatase is
a highly active enzyme, and even at very low quantity it can still induce
enough precipitation signals to be detected by a commercial digital CCD camera
mounted on a microscope (Fig.
2). Because the precipitate formation is stoichiometrical
to the amount of alkaline phosphatase, which again
strictly depends on that of miRNA captured by the
probe, the precipitate amount detected by CCD camera can be used to analyze
the miRNA level on each spot in microarray.
Microarray
fabrication
Eight
specific miRNAs from rat were chosen for a model miRNA
microarray system. The sequences of chosen miRNA
were obtained in Sanger database [2,21]. The eight target miRNA
sequences, the oligonucleotide probes, and the
negative control probe in the model microarray were listed in Table 1. The sequences of the eight oligonucleotide
probes are complementary to the corresponding target miRNAs.
All probes contain an amino modified 10-deoxyadenosines linker in the 5‘ terminal
for immobilization on the glass. The fabrication of the model miRNA
microarray was the same as described in our previous paper [14]. In brief, the probes were dissolved in 3´ SSC
(standard saline citrate) solution at
MiRNA labeling
In the
current method, the miRNAs to be detected require
labeling with biotin. The labeling was carried out as previously described [14]. Briefly, the enriched miRNAs of 90 mg from epididymis
of 9-week-old SD rat were dissolved in 30 ml diethylpyrocarbonate-treated
water and diluted with 10 ml of
Microarray
hybridization and detection
The
biotinylated miRNAs were dissolved in 4´ SSC, 0.1%
SDS, and hybridized with miRNA microarray as
previously described [14]. The hybridization reaction was performed at
45ºC overnight. And then the
hybridized microarray was washed with 1´ SSC/0.5%
SDS at 37ºC for 10 min. After this, the miRNA
level on the microarray was detected by either fluorescence or enzyme-linked
method. In the fluorescence method, the microarray was incubated with 10 ml of 2 nM streptavidin-conjugated
Qdot 655 (Invitrogen)
at room temperature for 1 h. After washing, the microarray was scanned on a ScanArray
5000 Scanner (PerkinElmer,
Data
analysis of microarray
The signal
intensity of miRNA microarrays
detected with the current method was expressed as the gray level of each spot
in the images obtained with CCD camera. In the data processing, the original
image from CCD camera was first transformed into gray-scale image, which was
further processed with the Photoshop 9.0 (Adobe System,
Results and Discussion
Figure
3 shows the feasibility of enzyme-linked detection method
application in miRNA microarray detection. The
inset image in Fig.
3 was obtained by the CCD camera as described in “Materials and Methods”.
The image shows that the concentration of model miRNA
probes spotted on the slides can be well detected by our method as expected in
schematic principle of the enzyme-linked detection method. The lowest detected
concentration is about Figure
4 shows the results obtained by enzyme-linked method to
detect the different concentrations of model miRNA.
The inset shows a set of images of various concentration miRNA
from 34 pM to 70 nM.
The image indicated that as low as 34 pM of miRNA
could be detected by the enzyme-linked detection method, which means the
detection limitation in this method was actually about 0.4 fmol
miRNA considering the printed volume of each spot
was about 1 nl. This detection sensitivity is very
close to that in fluorescence methods. It can be found in the image that the
signal intensities from lane 1 to 12 decreased gradually, showing an obvious
dependence on concentration of the model miRNA.
The plot in Fig. 4 indicated more clearly that the signal intensities were linear to the
logarithm of concentrations of model microRNA in
the range of 70 pM to 35 nM.
This suggests that the linear detection dynamic range in enzyme-linked method
was about 2.5 orders of magnitude. Such a detection sensitivity and broad
dynamic range can meet the needs for most miRNA
microarray detection, suggesting that the enzyme-linked detection method could
be used to detect miRNA microarray.
As known,
the signal intensity of enzyme reaction product is strictly time
dependent, the choice of the appropriate reaction time in enzyme-linked
detection method is very important. The optimum reaction time in the
microarray detection should satisfy two conditions. First, the reaction time
must be within the period when the enzyme reaction product is linearly
proportional to time. Second, the product amount produced within the reaction
time should give good signal to meet the detection requirement. Figure 5 indicated that the signal intensities were linear in the starting period of
about 100 min for 0.5, 5, and 50 nM biotin-labeled
model miRNA. The signal intensities reached a
plateau after 100 min. This phenomenon was even more obvious for higher
concentration of miRNA. Considering the signal
intensity and the time needed for whole enzyme-linked detection, we believe 40–60 min is the optimum time for enzyme reaction.
Figure
6 shows the results of detection and analysis of the model miRNA
microarray for eight miRNAs in rat obtained by
enzyme-linked detection method. The microarray contained four sub-arrays and
the printing pattern was also shown in Fig. 6. As shown in Fig.
6(A), the spots
corresponding to eight miRNAs displayed different
signal intensities, while the negative control was indistinguishable from the
background. Such signal pattern was similar to that of the image detected with
the fluorescence method shown in Fig. 6(B). The
quantitative analysis of the results from the two methods further indicated
that the average correlation coefficient of each miRNA
measured by both methods was about 0.93 [Fig. 6(D)].
The
reproducibility of the enzyme-linked detection method was shown in Fig. 7. The data in Fig.
7(A) indicated
that the error of each miRNA signal in different
sub-array was small. The correlation coefficients for these data in Fig. 7(A) were higher
than 0.96, showing a good reproducibility between four sub-arrays in a
microarray slides. The similar reproducibility between different slides was
shown in Fig. 7(B). The
correlation coefficients were 0.95, very close to that in the fluorescence
methods (about 0.97) in the same miRNA microarray
(data not shown). All these indicated that the enzyme-linked method is of high
reproducibility and reliable. Taken together, all the results mentioned above
indicated that the enzyme-linked detection method we established could provide
high detection sensitivity, large linear dynamic range and high
reproducibility in practical detection of miRNA
microarray. More importantly, this method has minimum instrumentation
requirement, only a commercial digital CCD camera and a microscope were
required. This method can reduce the threshold of miRNA
microarray experiment and is of potential applicability in miRNA
microarray and clinical diagnosis.
Funding
This work
was supported by the grants from the National Key Research and Development
Program of China (no. 2007CB935702) and the State Key Laboratory of Molecular
Biology of China.
References
1 Bartel DP. MicroRNAs:
genomics, biogenesis, mechanism, and function. Cell 2004, 116: 281–297.
2 Ambros V, Bartel
B, Bartel DP, Burge CB, Carrington JC, Chen X and Dreyfuss
G, et al. A uniform system for microRNA
annotation. RNA 2003, 9: 277–279.
3 Pasquinelli AE, Hunter S and Bracht
J. MicroRNAs: a developing story. Curr
Opin Genet Dev 2005, 15: 200–205.
4 Lee RC, Feinbaum RL and Ambros
V. The C. elegans heterochronic
gene lin-4 encodes small RNAs with antisense
complementarity to lin-14. Cell 1993, 75: 843–854.
5 Abrahante JE, Daul
AL, Li M, Volk ML, Tennessen JM, Miller EA and Rougvie
AE. The Caenorhabditis
elegans hunchback-like gene lin-57/ hbl-1 controls
developmental time and is regulated by microRNAs.
Dev Cell 2003, 4: 625–637.
6 7 Brennecke J, Hipfner
DR, Stark A, Russell RB and Cohen SM. bantam encodes a developmentally regulated microRNA
that controls cell proliferation and regulates the proapoptotic
gene hid in Drosophila. Cell 2003, 113: 25–36.
8 Iorio MV, Ferracin
M, Liu CG, Veronese A, Spizzo
R, Sabbioni S and Magri
E, et al. MicroRNA
gene expression deregulation in human breast cancer. Cancer Res
2005, 65: 7065–7070.
9 Zhao Y, Samal E and Srivastava
D. Serum response factor regulates a muscle-specific microRNA
that targets Hand2 during cardiogenesis. Nature
2005, 436: 214–220.
10 Calin GA, Liu CG, Sevignani
C, Ferracin M, Felli
N, Dumitru CD and Shimizu M, et al. MicroRNA
profiling reveals distinct signatures in B cell chronic lymphocytic
leukemias. Proc Natl Acad
Sci USA 2004, 101: 11755–11760.
11 Miska EA, Alvarez-Saavedra
E, Townsend M, Yoshii A, Sestan N, Rakic
P and Constantine-Paton M, et al. Microarray analysis of microRNA
expression in the developing mammalian brain. Genome Biol
2004, 5: R68.
12 Zhao JJ, Hua YJ, Sun DG, Meng
XX, Xiao HS and Ma X. Genome-wide microRNA
profiling in human fetal nervous tissues by oligonucleotide
microarray. Childs Nerv Syst
2006, 22: 1419–1425.
13 Zhao B, Liang R, Ge
L, Li W, Xiao H, Lin H and Ruan K, et al. Identification of
drought-induced microRNAs in rice. Biochem
Biophys Res Commun
2007, 354: 585–590.
14 Liang RQ, Li W, Li Y, Tan CY, Li JX, Jin YX
and Ruan KC. An oligonucleotide
microarray for microRNA expression analysis based
on labeling RNA with quantum dot and nanogold
probe. Nucleic Acids Res 2005, 33: e17.
15 Castoldi M, Benes
V, Hentze MW and Muckenthaler
MU. miChip:
a microarray platform for expression profiling of microRNAs
based on locked nucleic acid (LNA) oligonucleotide
capture probes. Methods 2007, 43: 146–152.
16 Thomson JM, Parker J, Perou
CM and 17 Beuvink I, Kolb FA, Budach
W, Garnier A, Lange J, Natt
F and Dengler U, et al. A novel microarray approach
reveals new tissue-specific signatures of known and predicted mammalian microRNAs.
Nucleic Acids Res 2007, 35: e52.
18 Liu CG, Calin GA, Meloon
B, Gamliel N, Sevignani
C, Ferracin M and Dumitru
CD, et al. An oligonucleotide
microchip for genome-wide microRNA profiling in
human and mouse tissues. Proc Natl Acad
Sci USA 2004, 101: 9740–9744.
19 Nelson PT, Baldwin DA, Scearce
LM, Oberholtzer JC, Tobias JW and Mourelatos
Z. Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 2004, 1: 155–161.
20 Joos TO, Schrenk
M, Hopfl P, Kroger K, Chowdhury
U, Stoll D and Schorner D, et al. A
microarray enzyme-linked immunosorbent assay for
autoimmune diagnostics. Electrophoresis 2000, 21: 2641–2650.
21 Griffiths-Jones S. The microRNA
registry. Nucleic Acids Res 2004, 32: D109–D111.
22 Liang RQ, Tan CY and Ruan
KC. Colorimetric detection of protein microarrays
based on nanogold probe coupled with silver
enhancement. J Immunol Methods 2004, 285: 157–163.
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