Real-time detection of mRNA splicing variants with specifically designed reverse-transcription loop-mediated isothermal amplification

Abstract

Alternative splicing is a ubiquitous and crucial process in cellular processes and has a specific linkage with diseases. To date, developing cost-effective methods with high sensitivity and specificity for detection of splicing variants has been needed. Herein, we report a novel splicing variant assay based on specifically designed reverse-transcription loop-mediated isothermal amplification. After reverse transcribing the splicing variant into cDNA, four DNA primers are specifically designed to recognize six distinct regions. The four DNA primers can hybridize with corresponding sequences for extension and strand displacement DNA synthesis to form stem-loop DNA and then LAMP amplification is started. The proposed method can detect as low as 100 aM splicing variants in real-time fashion with high specificity, showing great potential in biological function and clinical studies.

Fig. 1. Schematic representation of the proposed method for detection of mRNA splicing variants. The complementary sequences are indicated with solid and hollow lines, respectively. The 3′-end of the sequences are indicated with arrows.

Fig. 2. Detection sensitivity and dynamic range of the proposed method for FGFR 2 splicing variants. Real-time fluorescence curves were produced from different concentrations of FGFR 2-IIIc (a) and FGFR 2-IIIb (c). Linear relationship between POI values of the real-time fluorescence curves and the negative logarithm (−lg) of the FGFR 2-IIIc (b) and FGFR 2-IIIb (d) concentrations. The error bars in (b) and (d) were derived from three parallel experiments. NTC represents the reaction without the reverse transcription components and RNA target. Blank represents the reaction only without RNA target.

Fig. 3. Specificity of the LAMP-based method for FGFR 2 splicing variant detection. (a) Real-time fluorescence curves were originated from FGFR2-IIIc and FGFR2-IIIb with FGFR2-IIIc specific primers. (b) Real-time fluorescence curves were produced from FGFR2-IIIc and FGFR2-IIIb with FGFR2-IIIb specific primers.

Fig. 4. Quantification of FGFR 2 splicing variants in 100 ng total RNA from different cells. The ordinate was the copy number of the specific target in 100 ng total RNA and the abscissa indicated the cell lines. Error bars were calculated from three parallel experiments.