Methods for obtaining transcriptome data have revolutionized our understanding of biological processes and disease dynamics at the fundamental level, impacting health care on all three fronts: diagnostics, prognostics, and therapeutics. The compiling of transcriptome information, at one end of the spectrum leads to discovery of genes and mutations using, for example, the Sanger method and NetGen sequencing, and at the other end of the spectrum is the quantification of genetic expressions of known sequences using methods such as qPCR and microarrays. The proposed research pertains to the quantification of gene expression at few cell levels. Fundamental limitations of qPCR (the gold standard) and microarrays emerge from the inefficiencies and errors inherent to two necessary processes: cDNA synthesis by reverse transcriptase (RT) reaction, that requires more than 105 copies for reasonable efficiency, and subsequent PCR amplification, which may be prone to errors in exact replication. The goal of the proposed research is to develop a technology that eliminates these two processes and achieves at least an order of magnitude better sensitivity and better quality data in terms of self-consistency and normalization to accurately estimate relative expression levels of targeted mRNA. The expression level of 15 sequences from cell lines will be quantified simultaneously for this proof-of- principle study. The technology to be used is based on three principle steps: (i) targeting two unique sites of ~ 25 nucleotides on each mRNA of interest and exclusively separating the specifically bound target ssDNA sequences (TRID process); (ii) binding the ssDNA targets to a microarray mediated by electrochemical redox to obtain binding of ~ 1,800 molecules in 0.3 mL solution to microspots in less than 30 min at 100% specificity (EREB process); and (iii) reading the binding electrochemically at a responsivity of 0.4 zeptomole of probe- target binding to achieve a sensitivity of 10 attomolar and a dynamic range of five orders of magnitude (SEED process). It is expected that the time to result (TTR) after RNA extraction will be less than 3 hr. Specific Aim 1: Calibration of the SEED signal. The SEED signal for ssDNA targets for each mRNA from 10 aM to 10 pM in buffer will be measured to obtain calibration curves. The outcome will be the optimization of EREB to obtain copy numbers of all of the mRNA on a single chip. Specific Aim 2: qPCR study. Two cell lines of known dysregulation in genes will be cultured. The outcome will be mRNA expression of lysate of a known amount of cells by qPCR for all of the genes. Specific Aim 3: Technology verification study. TRID, EREB, and SEED will be performed at various dilutions of the same lysate solution used for qPCR. The outcome will be determination of the LOD and ENMC and the ability to measure at least 1.5-fold changes in copy number between the two cell lines and/or dilutions. Leveraging the high sensitivity and specificity, the long-term goal is to develop a quantitative microarray technology for gene expression of a few cells for applications such as single cell genomics, fine needle aspiration biopsy, and cell-free circulating nucleic acids.
Public Health Relevance Statement: A cell's function at the molecular level can be analyzed in great detail by measuring the expression level of mRNA which requires front-end processing, such as cDNA conversion and PCR amplification, that limits the sensitivity and accuracy. A microarray analysis using electrochemistry is proposed to measure binding as low as 0.4 zeptomole of the target to attain a sensitivity of 10 attomolar and will not require the abovementioned front-end processes. In the proposed proof-of-concept study, the expression of 15 mRNA will be quantified on a single chip from cell lines.
Project Terms: Address; base; Binding; Biological Process; Biology; Body Fluids; Buffers; Calibration; Cell Line; Cell physiology; Cells; Complementary DNA; Data; Data Quality; Detection; Diagnostic; Disease; Dyes; Electrochemistry; Electrodes; Expression Profiling; Fine needle aspiration biopsy; Fluorescence; gene discovery; Gene Expression; Gene Mutation; Genes; Genetic; Genomics; Goals; Gold; Healthcare; interest; invention; Kinetics; Label; Lasers; logarithm; Measurement; Measures; Mediating; Medicine; Messenger RNA; Methods; Methylene blue; Microarray Analysis; Molecular; mRNA Expression; Natural regeneration; Nature; Noise; Nucleic Acids; Nucleotides; Outcome; Oxidation-Reduction; Pattern; Performance; Phase; Process; prognostic; Reaction; Reading; Research; RNA; RNA-Directed DNA Polymerase; Scanning; Sensitivity and Specificity; Signal Transduction; Site; Specificity; Speed; Spottings; statistics; stem; success; Technology; Therapeutic; Time; transcriptome; Variant
