Micro RNAs (miRNAs) are short nucleotides (~ 20 nt) that act as regulators of gene expression in nearly all cellular processes including differentiatio, proliferation, and apoptosis. In tumors, miRNAs have been shown to play key roles in cancer processes such as metastasis and tumorigenesis. Since a single miRNA can regulate many mRNAs, dysregulation of one miRNA can have far-reaching biological consequences and that a small panel of miRNAs may suffice for diagnostic purposes. Recent discovery of the existence of circulating miRNAs in the blood stream further raises the potential of miRNAs as noninvasive biomarkers for remote cancer detection. In addition, due to their small size and protection within an exosomal shell, miRNAs robustly resist RNA degradation in tissue and blood. These features make miRNAs exciting targets for cancer diagnosis and prognosis. Since cell-free, circulating miRNAs exist at very low physiological concentrations, current methods to detect these targets predominantly rely on highly sensitive RT-qPCR. However, RT-qPCR is generally limited to single-plex analyses while clinical assessment of miRNAs requires that a panel (e.g.10-100) miRNAs to be quantified in a rapid and inexpensive manner. Employing a large number of separate PCRs for each sample is costly and requires large amounts of miRNA, which is difficult to obtain from blood samples. On the other hand, existing multiplexed technologies such as miRNA microarrays are woefully lacking in the requisite sensitivity to detect these circulating miRNA panels in body fluids. In this project, we propose to develop a single molecule length coding platform to address the unmet clinical need for highly sensitive and multiplexed detection of circulating miRNA. The platform employs a ligation- based molecular length coding scheme to generate miRNA-specific length-encoded strands that are deciphered by size separation to facilitate multiplexed detection. It utilizes cylindrical illumination confocal spectroscopy to quantify low concentration targets through single molecule counting, achieving high sensitivity and quantitative accuracy. In addition, a microfluidic device will be developed to simultaneously concentrate multiple microliter-sized samples into picoliter-sized plugs for arrayed separation in sub-micron channels to enhance both the resolution of separation and the throughput of analysis. Finally, we will validate the proposed platform by determining the analytical sensitivity and specificity using control serum samples spiked with synthetic miRNA sequences.
We aim to achieve PCR-equivalent sensitivity of <10-22 mole, specificity of >1000:1 for unrelated miRNA and >100:1 for related miRNA. Validation with clinical samples will be performed by analyzing a panel of 20 miRNAs in the serum of patients with advanced esophageal adenocarcinoma (20 samples) and healthy controls (20 samples) using d 200 mL of serum in a single reaction. The result will be compared to that obtained by RT-qPCR using 4 mL of serum split into 20 separate single-plex reactions.

Public Health Relevance

The goal of the proposed project is to develop a single-molecule length-coding platform capable of highly sensitive and multiplexed analysis of panels of miRNAs in the blood stream, addressing the unmet clinical need of using circulating miRNAs as cancer biomarkers.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA173390-02
Application #
8546323
Study Section
Special Emphasis Panel (ZCA1-SRLB-Q (O1))
Program Officer
Wang, Wendy
Project Start
2012-09-17
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
2
Fiscal Year
2013
Total Cost
$218,253
Indirect Cost
$83,529
Name
Johns Hopkins University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Huang, B; Song, J H; Cheng, Y et al. (2016) Long non-coding antisense RNA KRT7-AS is activated in gastric cancers and supports cancer cell progression by increasing KRT7 expression. Oncogene 35:4927-36
Friedrich, Sarah M; Liu, Kelvin J; Wang, Tza-Huei (2016) Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution. J Am Chem Soc 138:319-27
Friedrich, Sarah M; Zec, Helena C; Wang, Tza-Huei (2016) Analysis of single nucleic acid molecules in micro- and nano-fluidics. Lab Chip 16:790-811
Doucet-O'Hare, Tara T; Rodić, Nemanja; Sharma, Reema et al. (2015) LINE-1 expression and retrotransposition in Barrett's esophagus and esophageal carcinoma. Proc Natl Acad Sci U S A 112:E4894-900
Zhang, Xiaojing; Peng, Yin; Jin, Zhe et al. (2015) Integrated miRNA profiling and bioinformatics analyses reveal potential causative miRNAs in gastric adenocarcinoma. Oncotarget 6:32878-89
Rane, Tushar D; Zec, Helena C; Wang, Tza-Huei (2015) A barcode-free combinatorial screening platform for matrix metalloproteinase screening. Anal Chem 87:1950-6
Zhang, Yi; Wang, Tza-Huei (2015) High-resolution quantification by charge-dominant electrophoretic mobility shift of quantum dots. Electrophoresis 36:1011-5
Song, Yunke; Liu, Kelvin J; Wang, Tza-Huei (2015) Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1. Sci Rep 5:15620
Zec, Helena; Shin, Dong Jin; Wang, Tza-Huei (2014) Novel droplet platforms for the detection of disease biomarkers. Expert Rev Mol Diagn 14:787-801
Beh, Cyrus W; Pan, Deng; Lee, Jason et al. (2014) Direct interrogation of DNA content distribution in nanoparticles by a novel microfluidics-based single-particle analysis. Nano Lett 14:4729-35

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