MicroRNA detection specificity: the recent advances and future

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MicroRNA detection specificity: the recent advances and future perspective Tinglan Ouyang, Zhiyu Liu, Zhiyi Han, and Qinyu Ge Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05909 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Analytical Chemistry

MicroRNA Detection Specificity: the Recent Advances and Future Perspective Tinglan Ouyang, Zhiyu Liu, Zhiyi Han, and Qinyu Ge Abstract： MicroRNAs (miRNAs) are a series of promising molecules that could regulate gene expression, while its expression level and type are strictly related to the early diagnosis, targeted therapy and prognostic of diseases. Improved detection accuracy of miRNAs would lead to unprecedented progress in early treatment. Numerous methods to improve sensitivity have been proposed and confirmed valuable in miRNAs detection recently. However, the research which especially targets at improving detection specificity remains rare. This paper gave a retrospective summary of the most common detection methods including Northern blot, Reverse transcription quantitative polymerase chain reaction, Next-generation sequencing, and Microarray. Common methods play an important role in many aspects because of maturity and stability. However, the problem of specificity still exists. Then, this paper summarized breakthroughs in traditional techniques which are closely related to progressive methods to improve specificity. In addition, considerable studies focus on improving accuracy are described in detail. Most of them involve the reasonable combination of common methods, and the strategies that contribute to improving accuracy are classified. This paper aims to illustrate the importance of detection accuracy and explore how to improve specificity on detecting miRNAs, especially homologous families.

Keywords:

microRNA detection ； specificity ； selective structure ； strategies;

circulating miRNAs

Introduction MicroRNAs (miRNAs) are a series of non-coding small RNAs which is widely distributed in Eukaryotes (18-24nt), playing a role in silencing RNAs and regulating post-transcription negatively. 1 As emerging biomarkers, they participate in many ⁠

important physiological processes, including differentiation, apoptosis and the whole life cycle of cell growth. 2 Besides, since high-throughput sequencing has been widely ⁠

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spread, numerous studies have shown that the abnormal expression of miRNAs is associated with tumorigenesis. 3–7 The scientists and public have noticed that the ⁠

miRNAs may have a potential effect on RNA and other regulatory molecules. Herein, types of research have been done to profile their composition and expression level for understanding human physiological activities. Especially, miRNAs could be regarded as a series of promising molecules on cancer treatment, providing a novel scheme to diagnose and cure malignant illnesses as well as other pathologies. Cancer treatment, surrounded by a considerable number of targeted drugs and treatment strategies that have appeared in the market, is still a tricky problem. 8 Besides, patients are already in the middle or advanced stages of cancer when ⁠

they are in the hospital on most occasions. Even if patients are suffering from cancers, it is quite difficult to accomplish surgeries due to the expensive treatment cost and intrusive way. One encouragement is that the earlier detection of tumors and the identification of the correct subtype could greatly help most patients. Types of research surrounding the miRNAs expression profiles of normal, adjacent and tumor tissue, 9,10,6 ⁠

reveals the changes in biological signal molecules between the process of tumor development.11,12 An exciting thing based on the detection of miRNAs, the doctors have access to carry out effective gene therapy for victims and to estimate the risk of disease as early as possible, because diseases are easier to cure at the early stage. Another advantage is that the process to obtain the circulating tumor miRNAs is relative, not invasive compared to diseased tissue sampling. In order to better clarify the relationship between genotype and phenotype, it is necessary to study the role of abnormal expression of microRNAs in the pathological process. Consequently, the development of rapid, sensitive and quantitative circulating miRNAs detection platform and technology have attracted enough attention. When it comes to the requirements about what presented above, one of the most important indicators, sensitivity, has made abundant breakthroughs till now like decreasing the limit of detection using quantum dot13 and nanoparticles.14 However, it is difficult to apply the method from the research field to the medical clinical field where precise discrimination and quantification are essential simply considering the improvement of sensitivity. The specificity of the detection method is significant for miRNAs researches due to its high sequence homology which is closely related to the type, medication, and prognosis of related diseases. This paper reviews different methods applied in detecting miRNAs in recent years and summarizes promising strategies of miRNAs detection. The detection strategies are


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aimed at improving the specificity of circulating miRNA detection, which applies to all types of miRNAs. Besides, this article analyzes the detection discrimination of 1-4 base miRNAs experienced different methods and explores factors that affect the further improvement of specificity, including the number of base differences, the different position and bind energy in the nucleic acid chain.

Common detection methods Northern Blot Introduction ： Northern Blot is the most classic RNA detection method, including miRNAs, which is widely used till now. Based on molecular hybridization and gel electrophoresis, target molecules with different molecular weights run unequally under the electric field force, so that they reach different endpoints and formed some distinct electrophoretic strips. To a large extent, the reasons why it is considered to be the gold standard for RNA detection are its reliability and practicality. Advantages: After extraction, miRNAs are separated directly by denaturing gel electrophoresis. The following experimental procedures involve transfection, immobilization and hybridization ， which means the samples are not subjected to additional processing such as amplification. It is extremely reliable because there are no changes to the bases in the sequence, including modifications. Besides, the accuracy of Northern blot would be enhanced by complex and rigorous experimental preparation and electrophoresis systems, including whether the labors’ operation is rigid, the buffer is distributed uniformly, initial spotting positions are at same horizontal line, and how about the electrophoresis gel screening et.al. Furthermore, the sensitivity of Northern blot is closely related to specificity, and gels stripes cannot be displayed when limit of detection(LOD) is not reached. These stripes are not direct signals, but the differences from signal enrichment for different target molecules. Problems: Although this method was first discovered, there are still some problems. First, the entire experimental process is cumbersome, and sometimes the experimental conditions need to be optimized. Second, the required sample amount is relatively large which means the complex RNAs to be tested will consume a lot of time and materials. It is difficult for some clinical samples to meet this requirement such as circulating miRNAs. Third, it cannot distinguish miRNAs with different sequences from miRNAs with the same molecular weight. Two target molecules with different bases have exactly the same molecular weight, and this method will classify them as



one. Last but not least, it can only be qualitative and difficult to accurately quantify. The existing strategy to quantify is measuring the concentration of the cut-off gels, with the result that the operation is complicated and the difference may be indistinguishable, so it is more suitable for the detection of known miRNAs. Advances: In response to these problems, researchers have proposed improvements such as hybridization techniques, probe modification, and cross-linking methods (as Table 1 shown). Compared with other probes, modified oligonucleotide probe based on the lock nucleotide acid（LNA）improving the detection sensitivity nearly ten times. At the same time, the reduction in the number of mismatched probes indicates an increase in specificity.15 As shown in Figure 1, The processes of NB analysis typically include RNA separation, transfer to membrane and immobilization. The traditional method, immobilization using UV – Cross-linking is not suitable for small RNAs while applicable to RNAs longer than 70nt, besides the mechanism has not been elucidated yet. Therefore, Pall et al. researched another alternative method, 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemical modification to replace the original method, which modified miRNAs through 5 'phosphorylation reaction to maintain its stability.16,17 However, it remains radioactive, challenging to popularize and highly operational. Kim (2010) has developed a non-radioactive NB method to improve the practicability of the method.18,19 Huang (2014) analyzed the miRNA extracted from plants by using the probe of biotin markers, with a sensitivity of 5 µg and better long-term storage compared with the traditional probe. Summary: Although few of the latest literature reports due to its cumbersome and waste samples, Northern blot as a gold standard for detecting miRNAs remains irreplaceable.20–22 It can detect miRNAs themselves and other molecules that share the same sequence, such as precursors and mature miRNAs, which reflects the biological connections between molecules.23,24 However, when it comes to unknown target molecules, some coincidences are more likely to happen and lead to imprecise results. Therefore, the optimization around it is also worth exploring.

RT-qPCR Introduction: Reverse transcription quantitative polymerase chain reaction (RTqPCR) mainly includes the method which transcripts target miRNAs into cDNA reversely, then the cDNA is used as a template for real-time fluorescence qPCR. There is a linear relationship between the cycle threshold value (Ct value) of each template and the logarithm of the starting copy number of the template. When reaching the


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threshold, the number of miRNAs can be quantified. This technology almost balances the cost, accuracy, and sample amount,25,26 which also could be regarded as the “golden standard” for miRNAs profiling.27 Advantages: RT-qPCR is based on mature PCR technology and fluorescent probes, and the subsequent quantification process is highly automated. There are many developed instruments and commercial probes on the market, and the quality standards are relatively stable, which ensures the accuracy of the test. Another outstanding aspect of this method is the feasibility to integrate well with existing instruments like real-time PCR.28 Moreover, it could be used for absolute quantification which is not accessible to Northern blot. Problems: For small RNA, primer design of PCR is difficult because base pairing may be mismatched during amplification. When designing primers for the cDNA of miRNAs, the short length reduces the PCR efficiency because of low melting temperature,29,30 which means that if primer pairing area is consistent but the unpaired area is different, then false positives would occur. In clinical sample detection, it is more challenging and error-prone. Because it requires laborious sample preparation and rigorous experimental conditions, such as target molecule elongation (binding steps with extended sequences), miRNAs transcript into cDNA and amplification steps.31 In the optimization of experimental conditions, the activity of enzymes is affected by the high concentration of magnesium ions, and the inaccurate matching of random primers results in the deviation of results. In addition, PCR-based methods are also affected by bias in producing high-concentration amplifiers and the risk of sample contamination. Advances: Compared with qPCR, droplet digital PCR (ddPCR) is more sensitive and accurate in detecting cDNA.32 The development of ddPCR technology ensures that a single target molecule contained in a droplet. Then, the non-target genes can be removed through the detection of positive and negative fluorescence signals. While, the modified method combined the purification of multiplex PCR and microfluidics chip analysis, demonstrates primer has detrimental effects on miRNA profiling.33 Summary: In short, RT-qPCR is an established method, meanwhile sensitive and specific. However, the problem of sample-wasting still exist, requiring high purity samples so that the experimental operation cumbersome. What’s more, it can only be quantified and completely unable to recognize new expression profiles. Therefore, this method is often used to supplement or verify other methods.

High-throughput sequencing



Sequencing is widely used in the classification and analysis of many miRNAs, which can cover substantial genetic information. In addition to the ability to detect the expression of circulating miRNAs, and to determine the classification characteristics of the disease, new types of mutations can be found.34 Next-generation sequencing Many commercial Next-generation sequencing (NGS) machines have emerged, such as the Hi-Seq series. The base accuracy rate of the next-generation sequencing can reach 99.99%, and it is the preferred method in RNA expression analysis owing to a large amount of information. Not completely perfect, the period is long, and it is not suitable for quick clinical test. Besides, it still exits some bias-problems. Sequencing libraries are necessary for sequencing in the second generation, and PCR biasing issues arise during library preparation or sequencing. In the same system, high GC, secondary structure fragments are less likely to be amplified. In the sequencing results from the certain fragments, whether the number of repetitions is the actual reflection of the sample itself or the deviation caused by PCR is difficult to identify. However, it has not been explained clearly. Scientists speculate that is related to their energy differences. In summary, the Next-generation sequencing is not suitable for large-scale application in early diagnosis because of the long time and high cost, but it can detect unknown miRNA and identify new biomarkers massively and in parallel.

single-molecule sequencing The most important issue of third generation sequencing is the unsatisfied specificity. The accuracy of single-molecule sequencing, including technology mainly from Oxford nanopore and Pacific Biosystem, is not ideal. Nanopore mainly includes biological and solid. Bio-nanopores are easily denatured, modified or modified with membrane protein variants, which make them less convenient and cost-effective. It is difficult to find suitable wild-type nanopore, and the research is still shallow.35 Membrane stability and current noise need to be solved urgently. Solid-state nanopore depends entirely on artificial manufacturing technology. It is not easy to fabricate electrodes with regular shape and good electrical properties at the nanoscale. However, it could become the potential miRNA portable detection device with a combination of nanopore-isolated screening and nextgeneration sequencing.36 In general, the specificity of nanopore sequencing has


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encountered bottlenecks while it has a long read. PacBio sequencing captures four fluorescent-labeled nucleotides information during the replication process of the target molecules.37 This method also detects epigenetic modification information, such as base methylation. However, its initial detection error rate is as high as 20% while the subsequent sequencing cycle can increase the accuracy to 99.9% with the cost increasing.38 Because its sequencing principle is similar to the second-generation sequencing, which is based on the chemical reaction. So the error is accumulated, meanwhile, the read length is shorter than the nanopore sequencing.

Microarray Introduction: Microarray or biochip is a technology for rapid analysis and diagnosis by modifying the DNA probe on a solid surface, then the probe and the target of microRNAs interact to produce specific signals. Advantages: Compared with other detection methods, the microarray is of high throughput, high speed, multiplex, but can only detect known miRNA, moreover also has low sensitivity, more expensive and the defect of low selectivity. Problems: Microarray is mostly based on hybridization detection. The labeling of target is challenging because of the short probe target. In addition, the lower melting temperature of the duplex formed by the combination of probe and target significantly increased the risk of hybridization, resulting in mismatched sequences and false positive signals.39 Advances: A variety of ways to amplify the signal has been proven to significantly improve the sensitivity, such as the nanoparticles with good electrochemical performance directly amplified signal detection,40 utilizing enzymes to reduce chemical reaction activation energy for signal amplification,41 using digital PCR method reduce the background interference,42 etc. Summary: According to the above, although the sensitivity improvement is beneficial to the detection of the differences in the homologous miRNA signals, the specificity of the various methods for detecting the target molecule still lacks significant breakthrough. Therefore, the novel strategy to improve the specificity of miRNA detection worth exploring. Comparison of common methods



In addition, in situ hybridization (ISH) could identify target miRNAs within individual cells or in tissues while not common in circulating miRNAs. It has a lower concentration and easy to be destroyed. The ISH is rarely used for early diagnosis and detection. The descriptions of common methods for detecting small RNA are illustrated in table 2.

Selective strategies Through the above analysis of traditional methods and improved detection on microRNAs, it is found that the evaluation criteria for specificity for from useful. Some researchers report in their papers that their research strategies have high specificity only by comparing specific microRNAs with distinct sequences, limiting the connection with biological and clinical analysis. Some researchers regard the breakthrough on LOD as major progress on accuracy. It is true that the improvement of sensitivity plays a positive role in the improvement of specificity, but there are distinct evaluation indicators between the two. Here, we have selected a number of highly similar microRNAs detection studies from a wide range of literature, most of which come from homologous families or differ by 1-3 bases. By classifying these highly differentiated studies, the following three strategies are summarized. Based on the selective structure or biochemical reaction In terms of specificity, many methods hybridize the probe with the target RNA partly, resulting in some unmatched fragments. After combining with the designed nanoparticles or other probes used for amplification, they can be activated. If linking the probes and fluorescent signals, it is possible to use imaging techniques to distinguish the fluorescent signals. Due to the combination of DNA probes and RNA in the detection process, this hybrid chain that based combination often improves the diversity of homologous regions by designing selective structures or biochemical reactions, including hairpin structures or stem-loop structures, often known as molecular beacons.43–45 Stem-loop structure was first designed for real-time quantitative reverse transcription PCR.46 It is specific to the 3 'end of the mature miRNA and can expand the very short mature miRNA molecules, then utilizing a universal 3' primer site for real-time PCR. Compared with linear primers, stem-loop primers are high performance. The enthalpy variation between mature miRNA and precursor is more considerable,


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showing stronger stability. Base stacking can effectively extend the footprint of DNA/RNA duplex, and steric hindrance may impede its binding to double-stranded DNA. A microarray reaction on a carboxyl polyethylene glycol modified glass plate utilizes a hairpin structure as Figure 2. If the target miRNA exists, the hairpin structure opens and forms a duplex; if the capture probe is not opened, the golden nanoprobe conjugate signal probe will not be bound to the hairpin's stem area, thus preventing false positive signals. Then the gold nanoparticle labeled signal probe was combined with it.47 The detection limit of this method is 10fM, and the ability to distinguish the let-7a family is 74%-84%. Xueyuan Chen et al. （ 2018 ） have designed a molecular beacon as a capture probe and used lanthanide nano fluorescence to enhance and detect miRNA-21. Similar to the above principle, the capture probe can complement the target binding sequence and the detection probe, and then combine the nanoparticles with the biotin affinity and amplify the signal under the enhancement of the enhancer. It can show good practicability and early diagnosis ability in bovine serum solution with the complex background.48 The detection limit reached 1.38fM, and the division degree of the single base differential molecule was 52%, and the three base region division was 80%. The advantage of detection strategy based on the stem-loop structure for real-time PCR is that it can distinguish the only miRNA with a single base difference. The dynamic range of detection can be widely used for the detection of different concentrations, and the production of a novel structure is simple and quick. Another method that is considered to improve specificity is Poly A plus tail, proposed by Shi,R(2005).49 Because of the more straightforward preparation, many companies often use this strategy in their kits. RNA molecules without poly A will not be captured by magnetic beads, thereby eliminating the interference of nontarget miRNA. However, its distinction between precursors and mature miRNAs is not high, and it is difficult to detect 3 'terminal oxygen -2 methylation.50 In the above literature, structural design such as molecular beacons has significant discrimination for highly similar miRNAs. The advantage of the stem-loop primer method is that it is specific and economical. The disadvantage is that only one specific miRNA can be detected at a time, while the tailing method enables batch screening of miRNAs.



Based on system preparation and calculation The droplet digital PCR directly improves the sensitivity of the detection, and improves the purity to a certain extent, thereby enhancing the specificity. As shown in Figure 3, the probes A and B are complementary at the end, then hybridized and connected, and are assigned to water in oil droplets. When the positive signal is detected, the probe is cracked by splitting the tiny droplets containing a specific microRNA to produce a fluorescent signal, and the target miRNAs is directly condensed without the negative non-target molecules of the fluorescent signal. Inspired by the self-cleaning properties of titanium dioxide (TiO2), Wu and his colleagues (2018) developed a renewable super wet miRNA biochip (RSMB), which was used for the specificity and quantitative detection of miRNA-141. Superhydrophilic microspores were formed on superhydrophobic titanium dioxide substrates to form super hydrophilic super hydrophobic dual chips. First, an amino-functionalized capture probe is immobilized on the microspore. The sample solution containing target miRNA-141 and fluorescent labeling probe is added to the super-hydrophilic microspore simultaneously, then carry out the determination. Because of the wettability difference between the microspores and the substrates, the miRNA-141 and the detection probes are enriched and anchored to the microspores. Some of the miRNA141 bases are hybridized with the capture probes, and the other bases are hybridized with the detection probes to form the sand format. Therefore, fluorescent molecules are trapped on the microspores to produce fluorescent signals. However, no fluorescence signals were observed without microspores in miRNA-141. This attractive RSMB has enhanced sensitivity, long-term stability, and self-cleaning ability.51 This unique microporous may refer to the principle of ddPCR enrichment, but it establishes some more sophisticated microporous, unlike the tiny droplets. Jun Chen (2018) proved a novel method based on rational design and optimization of a closed symmetric dumbbell nucleic acid amplification template (STD template), which takes into account the free energy of different target molecules and designs a compact rigid template with standard free energy.52 As the figure 4 shown, it can ensure that only the target miRNA triggers the amplification reaction of the constant temperature index, meanwhile without the loss of amplification efficiency (the theoretical amplification efficiency is 2n), which effectively inhibits the non-specific hybridization and amplification. The detection limit of the method to let-7a is 0.01z mol. By detecting the homologous mixture under the complex background, it takes the let7a absorption value of the target molecule as the detection specificity. The selective


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precision is 3 bits after the decimal point, the formula of accuracy is calculated as: S% =

𝐴𝑙𝑒𝑡 ― 7𝑥 ― 𝐴𝑙𝑒𝑡 ― 7𝑎 1000𝐴𝑙𝑒𝑡 ― 7𝑎

× 100%

Johnsonbuck (2015) proposed a method to combine the target RNA with a biotinlabeled low noise capture probe, then the complex combined with a fluorescent probe on a streptomycin PEG-modified glass plate. The SiMRE method, inspired by the super-resolution imaging technology DNA-PAINT, has the most significant advantage in using the "reversible combination" mode of the DNA probe and the target miRNA, and the rate of the crosslink and the crosslink as the basis for qualitative and quantitative.53 Because of the difference in the rate of different nucleotides, and the binding process can be seen as a Poisson distribution, even if there is an even error, the correct bases in the four bases can be determined by many calculations of Poisson distribution. Sample preparation like ddPCR dramatically improves the purity of target miRNA and ensures that non-specific signal values are further reduced in small systems. On the other hand, regarding computational methods, the biological characteristics of the base sequence itself and the integration of mathematical laws may lead to a better breakthrough. Based on specific enzymes The enzyme assisted method utilizes the intrinsic sensing properties of various DNA / RNA reaction enzymes to match or mismatch substrates in specific regions.54 DNA connectivity analysis is based on high fidelity of DNA ligase.55 Jim J et al. (2016) described a simple, specific and sensitive miRNA detection method, using Chlorella virus DNA ligase (SplintRR Ligase). The two steps involve connecting the adjacent DNA oligonucleotides connected to miRNA, then analyzing target molecules by qPCR.56 The method detected the detection area of let-7b, Let-7c, and let-7g, and the electrophoresis pattern showed that the results had a significant degree of discrimination. Tomasz Krzywkowski and Mats Nilsson (2017) systematically explored the optimal conditions for RNA probe connection and detection. They proposed an iLock assay method by combining the structural specific cracking of the probe. After that, they use the Taq DNA polymerase and the PBCV-1 DNA ligase to improve the connection specificity. After rolling ring replication amplification and quantitative PCR and electrophoresis detection, the fidelity of this specific ligase for 3 'and 5' terminal



templates was measured. The ligase has the highest accuracy for the 5 'end T and A connection, reaching 92%; for C, the frequency of the mismatch base to T-C (40%) is even higher than the correct base pair G-C (34%) at the 3' end. In addition, except for the specificity of the enzyme, there is no additional modification of the nucleotide in the ilock detection method, which effectively reduces the non-specific reaction.57 Enzymatic amplification reduces the activation energy of molecular reaction and has high specificity in signal amplification and amplification. Therefore, screening and reforming enzymes with high fidelity is still the direction of development in the future.

Discussion and expectation The challenge of (circulating) miRNA for early rapid detection lies in low concentration and inaccurate typing, and the specific enhancement strategy can be combined with probe design, DNA-RNA system feature calculation, and microreaction system creation. At present, the common miRNAs detection methods include Northern blot, RTqPCR, sequencing, and biochip. When the effective target miRNA detection strategy is not yet established, the advantage of NB is obvious, so the improved NB is still worth studying. This will facilitate the popularization of small laboratories and testing methods. Meanwhile, RT-qPCR is also a gold standard for miRNA quantitative analysis. The accuracy of the Next-generation sequencing is very high, but expensive and not convenient, which is not suitable for Point-of-care Testing (POCT). Although the conventional techniques (Northern blot and RT-qPCR) are relative, not cost-effective and tedious to operate, these methods are widely used owing to their simple equipment and practical results. Nanopore sequencing can obtain all the sequence information of nucleic acid molecules, and its speed and sensitivity are fast, but its specificity is not high. The main causes are that the current signal is disturbed, the capture signal is discontinuous, and the nanopore fabrication process is not mature. Compared with the former, PacBio sequencing inherits the merits of SGS，which could arrive the higher accuracy after modification. However, it does not have the advantage of reads length. The microarray is easy to operate, and the results are clear and easy to get. It is very suitable for miRNA related screening and rapid detection of diseases, but it can only design corresponding beacons based on known miRNAs. This leads to a defect that the previous disease related miRNAs are typing may not be accurate and therefore


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requires more detailed classification of molecules. In traditional methods, the accuracy and simplicity of reverse transcription PCR are lacking, and the sensitivity of the developed ddPCR is significantly improved. The improved research of NB, such as LNA, EDC cross-linking, bioavidin labeling and so on, has also been applied to biochip-based and PCR-based detection. Besides, microarray chips with third-generation sequencing and multiple detection methods, represented by nanoscale sequencing, have prominent rapid detection advantages, and their selectivity and detection of composite samples are the directions of scientific research. In terms of base diversity, as table 3 shown, the diversity of the homologous sequence of three bases is 80%, and the focus of the breakthroughs is the single base and the double base difference sequence. Apart from the difference in base number, the specificity difference is also reflected in the base position. For the single base difference sequence, the base difference between the sequences is more easily distinguished,58 and the miRNA selectivity of the 3 ' is relatively low in relative position, which is different from the previous observation.59 Similar energy differences between bases may lead to nonspecific production, for example, the bond energies between T-A and T-G are almost the same.60 Besides, most of the detection methods rely on fluorescence detection, but autofluorescence also affects the detection results.61–63 A consensus has been reached on the use of homologous families for a specific comparison, but the accuracy of detection methods varies quantitatively. The detection and synthesis of miRNA in composite samples is difficult to compare parallel. Different detection methods involve different miRNA sequences, and even the same name does not necessarily distinguish between 3P and 5P, which leads to the difficulty of the method and the lower reliability. Therefore, it is necessary to establish a scientific and effective miRNA specific comparison standard.

Conclusion In conclusion, miRNAs possess good tissue stability as the promising marker for the disease. The establishment of their specific detection strategy is helpful to the classification of small molecule RNA so that they could be used in the treatment of diseases correctly and rationally. A single detection technique, whether it is NB, RTqPCR, microarray or sequencing, cannot meet the needs of accurate and rapid detection, and the appropriate detection methods often need to be integrated and utilized effectively by combining the previous detection strategies. The detection methods used



in the paper often use synthetic molecules hindering the connection with biological issues. Although good target data can be obtained, the plasma components in practical applications are involved. This suggests that the experiment should ensure that the higher purity of the extracted samples in addition to improving the specific strategy. some advancements have been achieved in the preparation methods such as ddPCR and microporous system. Stem-loop structure for improving selectivity has still been popular among many scholars. In microRNA detection accuracy, the brilliant future tends to be combined with the biochemical characteristics of the principle which considering the detection process and by means of statistical tools, rather than imprisoned in the traditional detection system.

Author Biographies Tinglan Ouyang and Zhiyu Liu are graduate students at Southeast University (SEU, China). Zhiyi Han and Qinyu Ge are partners in clinical research. Qinyu Ge is an assistant professor at SEU, China. The research interests of his group aim at RNA detection, genomics, and small DNA. Address correspondence about this article is [email protected].


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Table 1 Improved methods Based on Northern Blot References Anna Va ´lo ´czi (2004) Gurman S Pall(2008)

Sang Woo Kim(2010)

Qi Huang (2014)

Methods

LOD

Sensitivity

LNA3

2.5 ug

Middle

probe

EDC cross-linking DIG probe containing EDC and LNA biotin-labeled with UV cross

probes

30 fold than

Descriptions mismatched

probes

discrimination

Middle

Improved sensitivity

0.05 fM

High

Non-radioactive

5ug

Middle

Non-radioactive

UV-cross



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Table 2 Comparison of common detection Methods

Northern Blot

Specificity

High

Sensitivity

Middle

RT-qPCR

Middle

High

Microarray

Low

Low or Middle

Secondgeneration sequencing

Very High

Middle

Single molecular sequencing

Middle

High

A: advantages; D: disadvantages


Descriptions A: no-modification and established D:sample-consuming and tedious A: automatic quantification D:medium-throughput and cannot identify new miRNAs A ： low-cost, highthroughput，fast D：semi-quantitative， cannot identify new miRNAs A ： high-throughput and whole-genome D：expensive and longperiod A ： no-amplification and relative fast D：very expensive

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Table 3

Different strategies comparison to distinguish similar miRNAs

References

Methods

LOD

Liner range

Reference sequence

gold S. Roy （2016）

sequence homology discrimination

let-7b:~80%

nanoparticles

10fM

Microarray

10fM— 100nM

let-7a let-7f:~74%

with hairpin

let-7g:84% lanthanide Xue

yuan

Chen

（2018）

nanoprobes with

MB

1.38 fM

10fM-100 pM

miRNA-21

SM

：

52%

TM:80%

capture probe Renewable Li-Ping Xua（2018） superwettable

88 pM

biochip

0.1 nM to 50 nM

miRNA-21, miRNA-141

90%

SEXPAR with Jun Chen（2018）

rational

STD

0.01z M

NM

let-7a

NM

NM

let-7a

let-7 family over 92%*

design AJohnsonbuck(2015)

Kinetic fingerprinting qPCR

Jin, J（2016）

based

SplintRR

30 ag

Ligase

30 ag to 30 pg (miR-122)

miRNA-375 Over

let-7c 100%

miR-122 and the

let-7

obvious

family let-7c:81.7%

ligation Tian H(2016)

combined

20aM

ddPCR

20 aM to 200 fM

let-7e:94.4% let-7a

let-7b:96.7% let-7f:99.2% others: over 98.7%

MLPA P.Zhang(2013)

let-7c:75.11%

with

ribonucleotidemodified DNA

let-7b:96.48% 0.2fM

NM

probes

let-7a

let-7e:96.91% let-7d:99.75% others: over 99.9%



“NM” means not mentioned in this paper


Page 18 of 29

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Captions Fig. 1. Schematics for small RNA northern blotting protocol. EDC cross-linking facilitates the production of a covalent bond between the terminal phosphate of the RNA to a free amino group on the nylon membrane resulting in the small RNA becoming tethered by one end. (Reproduced with permission from ref.16. Copyright (2008) Nature Pub. Group).

Fig. 2. Schematic illustration of the miRNA detection assay on a microarray, which is produced on a carboxyl–PEG functionalized glass slide. (A) Immobilization of hairpin CPs. (B) CP–target hybridization at the loop region. (C) CP-SP hybridization at the stem region. (Reproduced with permission from ref.47. Copyright (2016) Elsevier Advanced Technology).

Fig. 3. Principle of the ddPCR-based miRNA assay. (Reproduced with permission from ref. 64. Copyright (2016) American Chemical Society).64 Fig. 4. Principle of the novel isothermal symmetric exponential amplification reaction. (Reproduced with permission from ref.52. Copyright (2017) American Chemical Society).



Figures

Fig. 1


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Fig. 2



Fig. 3


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Fig. 4



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A

AA

U

U

G

C

A G

C

5’

C C A

G

C

C C

G G

C G

G C U U UUUUU U

3’

Stem-loop structure

Electrophoresis

【EC】complex

specificity Quantum Dots

pre-microRNA

NGS

AuNP

sensitivity

3’

Raman

5’

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Microdroplets

MicroRNA detection specificity: the recent advances and future

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