Rapid mRNA Expression Analysis by Quantitative Electrochemical Microarray at Sub-Zeptomole Levels without PCR and Labels

Methods for obtaining transcriptome data have revolutionized our understanding of biological processes and disease dynamics at the fundamental levelimpacting health care on all three frontsdiagnosticsprognosticsand therapeuticsThe compiling of transcriptome informationat one end of the spectrum leads to discovery of genes and mutations usingfor examplethe Sanger method and NetGen sequencingand at the other end of the spectrum is the quantification of genetic expressions of known sequences using methods such as qPCR and microarraysThe proposed research pertains to the quantification of gene expression at few cell levelsFundamental limitations of qPCRthe gold standardand microarrays emerge from the inefficiencies and errors inherent to two necessary processescDNA synthesis by reverse transcriptaseRTreactionthat requires more thancopies for reasonable efficiencyand subsequent PCR amplificationwhich may be prone to errors in exact replicationThe 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 mRNAThe expression level ofsequences from cell lines will be quantified simultaneously for this proof ofprinciple studyThe technology to be used is based on three principle stepsitargeting two unique sites ofnucleotides on each mRNA of interest and exclusively separating the specifically bound target ssDNA sequencesTRID processiibinding the ssDNA targets to a microarray mediated by electrochemical redox to obtain binding ofmolecules inmL solution to microspots in less thanmin atspecificityEREB processandiiireading the binding electrochemically at a responsivity ofzeptomole of probetarget binding to achieve a sensitivity ofattomolar and a dynamic range of five orders of magnitudeSEED processIt is expected that the time to resultTTRafter RNA extraction will be less thanhrSpecific AimCalibration of the SEED signalThe SEED signal for ssDNA targets for each mRNA fromaM topM in buffer will be measured to obtain calibration curvesThe outcome will be the optimization of EREB to obtain copy numbers of all of the mRNA on a single chipSpecific AimqPCR studyTwo cell lines of known dysregulation in genes will be culturedThe outcome will be mRNA expression of lysate of a known amount of cells by qPCR for all of the genesSpecific AimTechnology verification studyTRIDEREBand SEED will be performed at various dilutions of the same lysate solution used for qPCRThe outcome will be determination of the LOD and ENMC and the ability to measure at leastfold changes in copy number between the two cell lines and or dilutionsLeveraging the high sensitivity and specificitythe long term goal is to develop a quantitative microarray technology for gene expression of a few cells for applications such as single cell genomicsfine needle aspiration biopsyand cell free circulating nucleic acids A cellandapos s function at the molecular level can be analyzed in great detail by measuring the expression level of mRNA which requires front end processingsuch as cDNA conversion and PCR amplificationthat limits the sensitivity and accuracyA microarray analysis using electrochemistry is proposed to measure binding as low aszeptomole of the target to attain a sensitivity ofattomolar and will not require the abovementioned front end processesIn the proposed proof of concept studythe expression ofmRNA will be quantified on a single chip from cell lines