Single-pair fluorescence resonance energy transfer analysis of mRNA transcripts for highly sensitive gene expression profiling in near real time.
Single-pair fluorescence resonance energy transfer analysis of mRNA transcripts for highly sensitive gene expression profiling in near real time.
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DOI：
10.1021/ac400729q
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发表时间：
2013-08-20
影响因子：
7.4
通讯作者：
Soper, Steven A.
中科院分区：
文献类型：
--
作者：
Peng, Zhiyong;Young, Brandon;Baird, Alison E.;Soper, Steven A.
从某些基因转录的mRNA的表达分析可以用作体外诊断的生物标志物的重要来源。虽然逆转录定量PCR（RT-qPCR）的使用可以为监测转录本数量提供出色的分析灵敏度，但许多关键应用需要更灵敏的表达分析方法，可以近实时报告结果。我们报告了一种新的检测方法，可以提供精致的定量限，包括逆转录（RT），然后连接酶检测反应（LDR）与单对荧光共振能量转移（spFRET），通过分子计数提供数字读数。对于该测定，不采用PCR，这使得测定周转时间短。为了促进测定的实施，采用使用热压印制造的环烯烃共聚物（COC）微芯片以连续流形式进行LDR，在LDR之后进行在线单分子检测。作为该测定的实用性的证明，从几种结直肠癌细胞系中表达MMP-7 mRNA。发现RT-LDR/spFRET测定即使在低拷贝数方案中也产生高度线性的校准图。与RT-qPCR的比较表明，在研究的低拷贝数范围（10 - 10，000个拷贝）内具有更好的线性，RT-LDR/spFRET的R2 = 0.9995，RT-qPCR的R2 = 0.98。此外，使用RT-LDR/spFRET可以以更高的置信度区分10和50的拷贝数。为了证明使用RT-LDR/spFRET测定可获得的短测定周转时间，对可用作缺血性中风的重要诊断标志物的两性霉素基因转录物进行2次热循环LDR。使用RT-LDR/spFRET在短的周转时间内提供关于可能的中风事件的诊断信息的能力将使临床医生能够用适当的时间敏感的疗法有效地治疗患者。
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.
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