Discriminative identification of miRNA let-7 family members with high specificity and sensitivity using rolling circle amplification

Abstract

Rolling circle amplification (RCA) is a new method based on virus DNA reproduction, which has been widely used in the field of miRNA detection. However, discrimination of highly homologous miRNAs is a bottleneck in the research of miRNA. In this study, the RCA process was creatively used to conduct the discrimination of miRNAs. Results showed that T4 RNA ligase 2 could reach the highest circularization efficiency during the RCA process with higher specificity. By using RCA technology, a member of highly homologous miRNAs, let-7, could be discriminated at the amount of 2.5 fmol. This sensitivity could not be achieved by using traditional reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. In addition, detection of miRNAs by using RCA could reach the amount limit of fmol with a good linearity. Optimal RCA technology used in this study is better than RT-qPCR in discriminating highly homologous family miRNAs. Results from this study can promote the applications of RCA in clinical diagnosis, environment protection, health care, disease inspection and prevention, and national security.

Bringing to Light the Importance of the miRNA Methylome in Colorectal Cancer Prognosis Through Electrochemical Bioplatforms

“…Moreover, considering the high homology between members of the miRNA let-7 family, 38 additional experiments comparing the amperometric responses provided by the bioplatform for the determination of total miRNA let-7a against two other homologous miRNAs from the same family (let-7f and let-7g, differing in one and two nucleotides as compared to let-7a, respectively) were performed. The results in Figure 3 a demonstrate that when using the shorter bCp-15, the miRNAs let-7f and let-7g gave 54 and 47% of the response provided by the target let-7a miRNA, respectively.…”

Parasite‐derived microRNA let‐7‐5p detection for metacestodiasis based on rolling circular amplification‐assisted CRISPR/Cas9

“…30,31 Moreover, qPCR cannot discriminate against highly homologous miRNAs. 32 Other methods, such as northern blotting and microarray assays, suffer let-7-5p gradually increased in the plasma of T. pisiformis-infected rabbits from 15 days post infection (dpi), peaked at 60 dpi, and persisted until 120 dpi. In E. multilocularis-infected mice, let-7-5p gradually increased from 15 dpi and persisted until 90 dpi.…”

“…For instance, qPCR is time‐consuming and requires multiple steps, including primer design, miRNA extraction, reverse transcription, and qPCR itself 30,31 . Moreover, qPCR cannot discriminate against highly homologous miRNAs 32 . Other methods, such as northern blotting and microarray assays, suffer from poor detection specificity and sensitivity, lengthy processes, and high detection costs 33,34 .…”

Rolling Circle and Loop Mediated Isothermal Amplification Strategy for Ultrasensitive miRNA Detection

“…In recent years, some new technologies, such as assays based on isothermal nucleic acid amplification, have been applied to detect miRNAs [6][7][8][9][10][11][12][13][14]. Among them, rolling circle amplification (RCA) has become more popular, owing to its specificity and simplicity [7,[13][14][15][16][17][18][19][20][21][22][23][24]. The miRNA always serves as a ligation template, which is hybridized with the padlock probe.…”

“…Then, this DNA complex will be ligated by an enzyme to form a circular single-stranded DNA (ssDNA). An external primer or miRNA itself works as primer to extend on the circular ssDNA, and eventually displace the linked miRNA, then long cascaded products, which are the repeated sequences from circular ssDNA, are produced [14,15,[17][18][19][22][23][24][25][26][27]. However, the detecting of RCA products is exactly laborious and time-consuming, although several kinds of methods based on RCA were designed for simple and specific miRNA expression analysis including MNAzyme-mediated [13],…”