Multiplex Ligation-dependent Probe Amplification (MLPA) For Sensitively Multiplexed MicroRNA Detection

MicroRNAs (miRNAs) are a class of endogenous, non-protein coding and small RNAmolecules that play important regulatory roles in gene expression by the formation of anRNA-induced silencing complex (RISC) with target mRNA. Recently, miRNAs have showngreat importance in regulating a wide variety of biological processes including cellproliferation, differentiation, immunological response and tumorigenesis. Many studies haveshown that miRNA-expression alterations in a specific cancer tissue generally involvedseveral miRNA species (typically2-15miRNAs). Therefore, sensitive, accurate andmultiplexed miRNA detection has great significance for cancer diagnosis, as well as for betterunderstanding of the mechanism of tumorigenesis.In this paper, we demonstrated that the DNA probe modified with two ribonucleotidescan be efficiently and specifically ligated with T4RNA ligase2templated by miRNAs basedon multiplex ligation-dependent probe amplification (MLPA). With PCR amplificationfollowed by capillary electrophoresis, as low as0.2fmol/L target miRNA can be accuratelydetermined and a single-base difference among miRNA sequences can be discriminate withhigh specificity. To test the multiplexed ability of the assay, we randomly chose mir-92a,mir-31, mir-143, mir-145and let-7a which sequences are unrelated to each other. By codingthe different miRNAs with different length of DNA probes, all5kinds of miRNAs can besensitively detected within one-tube PCR amplification. We also applied our MLPA-basedmiRNA detection assay to detect miRNAs in total RNA extracted from A549cell line; all5kinds of miRNAs can be well detected in the total RNA sample, indicating the proposed assaycan be sucessfully applied to miRNA detection in the real samples. Therefore theMLPA-based multiplex miRNA detection assay would provide a useful tool for robust,ultrasensitive and multiplex quantification of miRNAs.