Abstract
Rolling circle amplification (RCA) and loop mediated isothermal amplification (LAMP) were combined to establish the rolling circle and loop mediated isothermal amplification (RC-LAMP) method for miRNA detection. With the participation of Bst 2.0 DNA Polymerase, the method enabled RCA and LAMP amplification to occur simultaneously without thermal cycling. The limit of detection of RC-LAMP was 500 amol/L, which is comparable to previously reported amplification strategies. Moreover, its upper limit of quantitation was higher and showed a stronger resistance to matrix interference. Therefore, it is possible to detect low concentrations of miRNA in samples by increasing the total RNA added. Owing to its facile detection mode and simple operation, this method has great potential in clinical sample detection.
Highlights
Amplification Strategy for MicroRNAs are small, non-coding RNAs with 19–24 nucleotides, which regulate two thirds of the protein-coding genes in humans [1]
The schematic representation of miRNA detection by RC-loop mediated isothermal amplification (LAMP) method is shown in Scheme 1
The backward inner primer (BIP) which contains B2 and B1c (B1 complementary) sequences bind to the B2c (B2 complementary) sequence on the circular single-stranded DNA (ssDNA) to initiate the Rolling circle amplification (RCA) reaction under the action of Bst 2.0 DNA Polymerase with strand displacement activity, creating a single-stranded repeat sequence
Summary
Amplification Strategy for MicroRNAs (miRNAs) are small, non-coding RNAs with 19–24 nucleotides, which regulate two thirds of the protein-coding genes in humans [1]. MiRNAs have been confirmed to be involved in a variety of cellular functions and biological and pathological processes, including the expansion, differentiation, metabolism and apoptosis of cancer cells [2]. As a regulator of many cancer-related genes, miRNAs are expected to become biomarkers for the diagnosis and prognosis of cancer [3]. A miRNA has the functions of binding to functional proteins, regulating mitochondria-related gene mRNAs, directly activating gene transcription and targeting other non-coding RNAs [4]. Based on the above biological functions, miRNAs become a promising therapeutic drug in a variety of pathological processes. It is imperative to establish sensitive and selective miRNA detection strategies for the sake of medical applications or to understand their functions
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