A Reverse Transcription-free Assay for Gene Expression Analysis of Highly Degraded mRNA Samples

To accurately determine such highly degraded mRNAs, a reverse transcription. free assay for gene expression analysis based on a ligation reaction was proposed. In this method, a pair of probes with target-specific sequences complementary to a short mRNA fragment was employed. Once hybridizing to a target, the probes could be ligated by the ligation reaction, generating the template for the followed real-time quantitative PCR (qPCR) detection. To investigate the sensitivity and the specificity of the method, a pair of probes targeting human ACTB gene (beta-actin) was designed to detect a synthetic RNA template with different concentrations and another RNA template which was non-complementary to the probes. The proposed method was used to detect expression levels of ACTB gene in formalin-fixed and paraffin-embedded tissue sections of lung cancer, and the accuracy of the method was compared with that of reverse-transcription qPCR. The results showed that the detection limit of this method was 150 fmol/L with a dynamic linear range of 300 pmol/L - 150 fmol/L, and the method showed an excellent specificity. In terms of analyzing gene expression of paraffin-embedded samples, our method achieved a lower CT value than that of reverse-transcription qPCR, indicating that our method was more suitable for the gene expression determination in highly degraded mRNA samples.