Isothermal Amplification for MicroRNA Detection: From the Test Tube to the Cell

Abstract

MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as pivotal post-transcriptional regulators of gene expression, thus involving in many fundamental cellular processes such as cell proliferation, migration, and canceration. The detection of miRNAs has attracted significant interest, as abnormal miRNA expression is identified to contribute to serious human diseases such as cancers. Particularly, miRNAs in peripheral blood have recently been recognized as important biomarkers potential for liquid biopsy. Furthermore, as miRNAs are expressed heterogeneously in different cells, investigations into single-cell miRNA expression will be of great value for resolving miRNA-mediated regulatory circuits and the complexity and heterogeneity of miRNA-related diseases. Thus, the development of miRNA detection methods, especially for complex clinic samples and single cells is in great demand. In this Account, we will present recent progress in the design and application of isothermal amplification enabling miRNA detection transition from the test tube to the clinical sample and single cell, which will significantly advance our knowledge of miRNA functions and disease associations, as well as its translation in clinical diagnostics. miRNAs present a huge challenge in detection because of their extremely short length (∼22 nucleotides) and sequence homology (even with only single-nucleotide variation). The conventional golden method for nucleic acid detection, quantitative PCR (qPCR), is not amenable to directly detecting short RNAs and hardly enables distinguishing between miRNA family members with very similar sequences. Alternatively, isothermal amplification has emerged as a powerful method for quantification of nucleic acids and attracts broad interest for utilization in developing miRNA assays. Compared to PCR, isothermal amplification can be performed without precise control of temperature cycling and is well fit for detecting short RNA or DNA. We and other groups are seeking methods based on isothermal amplification for detecting miRNA with high specificity (single-nucleotide resolution) and sensitivity (detection limit reaching femtomolar or even attomolar level). These methods have recently been demonstrated to quantify miRNA in clinical samples (tissues, serum, and plasma). Remarkably, attributed to the mild reaction conditions, isothermal amplification can be performed inside cells, which has recently enabled miRNA detection in single cells. The localized in situ amplification even enables imaging of miRNA at the single-molecule level. The single-cell miRNA profiling data clearly shows that genetically identical cells exhibit significant cell-to-cell variation in miRNA expression. The leap of miRNA detection achievements will significantly contribute to its full clinical adoption and translation and give us new insights into miRNA cellular functions and disease associations.

Nucleic acid aptamer-based methods for diagnosis of infections

“…3. Readers are referred to recent reviews for a more comprehensive overview of isothermal nucleic acid amplification techniques (Bi et al, 2017;Deng et al, 2017;Giuffrida and Spoto, 2017;Zhao et al, 2015). (A) An aptamer-based fluorescence assay for the detection of S. Typhimurium using aptamers either modified with a fluorescent label (FAM) or linked to magnetic nanoparticles (Duan et al, 2013).…”

In Situ Amplification‐Based Imaging of RNA in Living Cells

“…[40] In RT-PCR, atarget RNAofinterest is first reverse transcribed into complementary DNA(cDNA) transcripts by reverse transcriptase.T hen, the newly-produced cDNAi s amplified through traditional PCR in the presence of thermostable polymerase,r esulting in indirect amplification-based detection of the target RNA. [44][45][46][47][48][49][50][51][52][53] In FISH, enzymes that can catalyze the amplification of nucleic acid under ambient temperature are generally transferred into cells,t hen the target nucleic acid of interest is amplified, leading to af luorescence signal. [41][42][43] In addition, isothermal enzymatic signal amplificationbased fluorescence in situ hybridization (FISH) assay has been developed for single-cell RNAi maging.…”

“…[59,60] Therefore,developing effective signal amplification strategies that can operate inside live cells [61] is the key to in situ amplified monitoring of intracellular RNAa tl ow copy numbers. [44,52,[62][63][64] 3. Non-enzymatic and Isothermal Signal Amplification Techniques Dynamic DNAs elf-assembly (DDSA) is fueled by the free energy of hybridization between metastable DNAh airpins or driven by the entropy of hybridization between linear DNAm olecules, [63,[65][66][67][68][69][70][71][72][73] and can operate in an autonomous and reconfigurable manner.F or instance,t he hybridization chain reaction (HCR) was first proposed by the Pierce group in 2004 ( Figure 1A).…”

“…[41][42][43] In addition, isothermal enzymatic signal amplificationbased fluorescence in situ hybridization (FISH) assay has been developed for single-cell RNAi maging. [44][45][46][47][48][49][50][51][52][53] In FISH, enzymes that can catalyze the amplification of nucleic acid under ambient temperature are generally transferred into cells,t hen the target nucleic acid of interest is amplified, leading to af luorescence signal. Fore xample,t he Li group developed an assay,t ermed toehold-initiated rolling circle amplification (TIRCA), for visualizing individual miRNAs in situ in single cells by transferring Phi 29 DNApolymerase Owing to its important physiological functions,e specially as molecular biomarkers of diseases,R NA is an important focus of biomedicine and biochemical sensing.Signal amplification detection has been put forward because of the need for accurate identification of RNAat low expression levels,w hichiss ignificant for the early diagnosis and therapyo fmalignant diseases.H owever,conventional amplification methods for RNAa nalysis depend on the use of enzymes,f ixation of cells,and thermal cycling,whichc onfine their performance to cell lysates or dead cells,thus the imaging of RNAinliving cells remained until recently little explored.…”

Progress in miRNA Detection Using Graphene Material–Based Biosensors

“…Although in the past several years, several review articles summarizing miRNA detection approaches have been published, to the best of our knowledge, a review article focusing on graphene‐based miRNA systems is still not available. Herein, this review article critically and comprehensively summarizes the recent progress in miRNA detection based on graphene‐based materials, with an emphasis on i) the underlying working principles of these types of sensors and the unique roles and advantages of the graphene materials used; ii) state‐of‐the‐art protocols developed for high‐performance miRNA sensing, including electrochemical detection methods and fluorescence methods, with representative examples; iii) future perspectives and the current challenges of graphene‐based sensors.…”