Single-Pair Fluorescence Resonance Energy Transfer Analysis of mRNA Transcripts for Highly Sensitive Gene Expression Profiling in Near Real Time

Abstract

Expression analysis of mRNAs transcribed from certain genes can be used as important sources of biomarkers for in vitro diagnostics. While the use of reverse transcription quantitative PCR (RT-qPCR) can provide excellent analytical sensitivity for monitoring transcript numbers, more sensitive approaches for expression analysis that can report results in near real-time are needed for many critical applications. We report a novel assay that can provide exquisite limits-of-quantitation and consists of reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) to provide digital readout through molecular counting. For this assay, no PCR was employed, which enabled short assay turnaround times. To facilitate implementation of the assay, a cyclic olefin copolymer (COC) microchip, which was fabricated using hot embossing, was employed to carry out the LDR in a continuous flow format with on-line single-molecule detection following the LDR. As demonstrators of the assay's utility, MMP-7 mRNA was expression profiled from several colorectal cancer cell lines. It was found that the RT-LDR/spFRET assay produced highly linear calibration plots even in the low copy number regime. Comparison to RT-qPCR indicated a better linearity over the low copy number range investigated (10 − 10,000 copies) with an R2 = 0.9995 for RT-LDR/spFRET and R2 = 0.98 for RT-qPCR. In addition, differentiating between copy numbers of 10 and 50 could be performed with higher confidence using RT-LDR/spFRET. To demonstrate the short assay turnaround times obtainable using the RT-LDR/spFRET assay, a 2 thermal cycle LDR was carried out on amphiphysin gene transcripts that can serve as important diagnostic markers for ischemic stroke. The ability to supply diagnostic information on possible stroke events in short turnaround times using RT-LDR/spFRET will enable clinicians to treat patients effectively with appropriate time-sensitive therapeutics.

Recent and near-future advances in nucleic acid-based diagnosis of stroke

“…qPCR-based approaches are more likely than microarrays to be applied and developed for rapid assays and automated POC systems that would be needed for early stroke diagnosis [62,63]. Compared with microarrays, qPCR approaches are characterized by shorter assay turnaround times and significantly higher sensitivity.…”

“…The assay uses RT-LDR/spFRET to digitally count and quantify mRNAs (i.e., molecular counting) for securing exquisite limits-of-quantitation in the low copy number regime [63]. Because single molecules are counted, the assay also does not require calibration curves.…”

“…Therefore, the MB adopts a stable stem-loop conformation following ligation forming the MB. The spFRET signal from individual MBs is reported by a SMD instrument from which the copy number of the mRNA could be deduced [63].…”

“…The LDR/spFRET assay can be spatially multiplexed using a wide-field CCD imaging microscope and microfluidics [63]. For 30 mm wide microfluidic channels, the multiplexing power (the number of different mRNAs that can be detected in a single run) would be 30 [80].…”

Recent Advances in Applications of Droplet Microfluidics

“…The reagent use can be reduced to nanoliters or less, and the reaction time can be reduced to mere seconds. Miniaturizing biological assays or processes on a chip has emerged as a hopeful technology [3][4][5][6][7].…”