| Gene expression analysis is one of the conventional methods for biological and medical research. It has been widely used in studies of gene function, gene interaction and personalized medicine. Recent years have witnessed the emergence of many gene expression measurement methods, such as northern blot, FISH, microarray, real-time PCR and sequencing. Among them, real-time PCR, as the gold standard for gene expression analysis, have been utilized by lots of researchers because of its high sensitivity, good specificity, excellent quantitative performance and simple operation. However, in the clinical research, it is usually required to determine the gene expression in highly degraded samples such as FFPE. Since the mRNA in these samples are highly degraded, their quantification is hard to achieve by traditional gene expression measurement assays, including real-time PCR. Thus we really need to develop a gene expression method for degraded mRNA samples,In order to solve the problems above, a reverse transcription-free assay is proposed for gene expression analysis based on ligation reactions. First, a pair of specific probes with primer sequences was designed. Once hybridizing to the mRNA template, the probes will be ligated by ligase, so that generate a DNA chain. The DNA chain will be detected the following real-time PCR. In this study, we have optimized the conditions such as temperature, concentration of probes and amount of ligase. We have also investigated the sensitivity, specificity, quantitative performance of the proposed method, then compared it to reverse-transcription real-time PCR in the analysis of highly degraded samples. As a result, the sensitivity of our method is 150 fmol/L, and the specificity is excellent. Moreover, the dynamic linear range is from 300 pmol/L to 150 finol/L, thus being able to determine 20-30 nt mRNA. In conclusion, this method is an accurate gene expression analysis for highly degraded samples, providing a powerful tool for life sciences. |
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