Highly sensitive detection of microRNA by chemiluminescence based on enzymatic polymerization
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- Ma, C., Yeung, E.S., Qi, S. et al. Anal Bioanal Chem (2012) 402: 2217. doi:10.1007/s00216-011-5653-4
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We have developed a new methodology for miRNA assay using chemiluminescence imaging by poly(U) polymerase catalyzed miRNA polymerization. This method is very sensitive with a 50 fM limit of detection, which is comparable to or better than current assay methods. Multiplex detection for miRNA can be easily realized by introducing different capture probes onto the biosensor array, which will make it highly versatile for various research purposes.
MicroRNA (miRNAs) is a class of small (approximately 19 to 23 nucleotides), endogenous, noncoding RNAs that are powerful transcriptional and post-transcriptional regulators of gene expression and cell development in animals, plants, and viruses [1–4]. Specific changes in miRNA expression levels are associated with a variety of diseases, including cancer [5, 6], neurodegenerative disorders , diabetes , and represents promising biomarker candidates for early cancer diagnosis . For example, the highly tissue and developmental stage-specific expression of miRNA allows for the accurate molecular classification of tumors . Therefore, detection of miRNAs has great significance not only for understanding their biological functions but also for clinical diagnosis of human diseases as well as the discovery of new drugs through the use of miRNAs as targets [11, 12]. The most widely reported method for miRNA detection, Northern blotting, requires substantial amounts of starting material, is extremely laborious, and is not sensitive [13–15]. Recently, a number of new methods for microRNA detection have been reported, such as electrochemistry [16–20], fluorescence [21–24], capillary electrophoresis [25, 26], surface plasmon resonance (SPR) imaging [27, 28], and atomic force microscopy . These reported methods have high sensitivity, but often at the expense of assay simplicity, multiplexing capability, or rapid analysis time.
Luciferase from firefly Photinus pyralis, d-luciferin, adenosine phosphosulfate (APS), adenosine triphosphate sulfurylase from Saccharomyces (ATPase), 3-aminopropyltriethoxysilane (APTES), and glutaraldehyde were purchased from Sigma-Aldrich (St. Louis, MO). The 1× Hanks balanced salt solution (HBSS), and uridine triphosphate (UTP) were purchased from Life Technologies (Carlsbad, CA). Poly(U) polymerase (2,000 units/mL) was purchased from New England Biolabs (NEB, U.K.). 1× phosphate-buffered saline (PBS) buffer (pH 7.4) was purchased from Life Technologies. PBS (20×) Tween-20 buffer was purchased from Pierce Protein Research Products-Thermo Fisher Scientific (Rockford, IL). Ultrapure water from a Milli-Q system (Millipore, Billerica, MA) was used throughout the experiments. All chemical reagents were used without further purification.
DNA and RNA oligonucleotides
All 3′-amino-modified LNA-DNA oligonucleotide probes (Exiqon, MA) were purified using HPLC. The amino-modified LNA probes used were: miR-20 LNA = 5′-CACTATA AGCACTTT ATTTTTTTT-3′, and let-7a LNA = ACAACCTACTACCTC ATTTTTTTT-3′ (all LNA bases are underlined). All synthetic miRNA sequences used were purchased from TriLink Biotechnologies (San Diego, CA) and purified using HPLC. The miRNA sequences used in this work are: miR-20 = 5′-UAAAGUGCUUAUAGUG CAGGUA-3′, let-7a = 5′-UGAGGU AGUAGGUU GUAUAGUU-3′.
Surface derivatization and LNA probe immobilization
Cover slips with dimensions of 22 × 22 mm (Electron Microscopy Sciences, Hatfield, PA) were cleaned in an ultrasonic bath for 15 min in detergent and ultrapure water, 15 min in ultrapure water, and 15 min in methanol (twice). After cleaning and surface treatment, cover slips were silanated in a 1% (v/v) APTES-ethanol solution for 1 h under agitation. Next, the APTES surfaces were activated with 2% (v/v) glutaraldehyde in 1× PBS at pH 7.4 for 1 h. Amino-modified LNA probe at a concentration of 200 pM in 1× PBS was then incubated onto the activated surface for 2 h. The cover slips were then washed with 1× PBS. The remaining amine-active sites were blocked with 1% BSA solution for 1 h, and then washed with PBS Tween-20 buffer.
Detection of miRNA by chemiluminescence
(a) miRNA Hybridization with LNA Probe. Reactions were performed in a volume of 20 μL in a 1× HBSS buffer solution at 37 °C for 30 min. The cover slip was then rinsed with 1× HBSS buffer containing 10 mM MgCl2 for 3 min. (b) Poly(U) Polymerase Reaction. After miRNA hybridization, the cover slip was reacted with a mixture of poly(U) polymerase (0.2 units/μL) and 1 mM UTP in a volume of 15 μL in a reaction buffer of 10 mM Tris–HCl, 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT (pH: 7.9) for 30 min. (c) PPi Conversion and Bioluminometric Assay. The reaction product PPi was converted to ATP for 10 min by injection of 15 μM APS, and 0.05 unit ATP sulfurylase into the existing buffer. Finally, 1 mM d-luciferin and 1.67 μM luciferase reaction solution was injected in a final volume of 20 μL, and the bioluminescence signal was measured with chemiluminescence microscopy.
The imaging system consists of an inverted light microscope (Nikon Diaphot 300, Fryer, Edina, MN) and a complex electron-multiplying microchannel plate coupled ICCD (EEV 576 × 384, Princeton Instruments, Trenton, NJ) attached to the camera mount of the microscope. The ICCD camera was operated at −30 °C and read out at 430 kHz with 12-bit resolution. A 100× oil immersion objective (N/A 1.25, Nikon) was used for miRNA concentration detection, and a 10× plano objective lens (Mitutoyo, Japan) was used for miRNA array study. The whole system was placed in a dark box. WinView 32 (Roper Scientific, Princeton, NJ) was used for image collection, and NIH ImageJ was used to analyze and process the collected images.
Results and discussion
Effect of poly(U) polymerase reaction time on miRNA detection
Detection of miRNA by chemiluminescence
Chemiluminescence array detection of miRNA
In summary, we have developed a new methodology for miRNA assay using chemiluminescence imaging by poly(U) polymerase catalyzed miRNA polymerization. This method is very sensitive with a 50 fM limit of detection, which is comparable to or better than current assay methods. Multiplex detection for miRNA can be easily realized by introducing different capture probes onto the biosensor array, which will make it highly versatile for various research purposes. Future efforts will be directed towards increasing the levels of multiplexing with microarray spotting technologies for the rapid encoding of many unique sensing targets. For example, the size of the probe spot could be reduced by perhaps 20-fold (see Fig. 4) to allow a higher density array and to accommodate smaller sample amounts.
The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. DE-AC02-07CH11358. This work was supported by the Director of Science, Office of Basic Energy Science, Division of Chemical Sciences.