Amplification-free gene expression analysis of formalin-fixed paraffin-embedded samples using scanning single-molecule counting

Abstract

In this paper, we present the applications of our newly developed, highly sensitive fluorescent detection method referred to as scanning single-molecule counting (SSMC). We found that the target RNA added to the total RNA was detected with high sensitivity at 384 aM by combining a magnetic bead-based assay and SSMC (MB-BA + SSMC). Gene expression analysis without reverse transcription or amplification confirmed that the pattern of gene expression was identical to that of real-time polymerase chain reaction (PCR). MB-BA + SSMC was also applied to formalin-fixed paraffin-embedded (FFPE) samples. RNA fragmentation and crosslinking owing to FFPE processing slightly affected gene expression. Conversely, FFPE samples showed an increase in gene Ct values and a decrease in the number of detectable genes when analyzed using real-time PCR. Overall, our results suggested that SSMC is a powerful tool for target RNA detection and amplification-free gene expression analysis.