PAXgene-fixed paraffin-embedded sample is applicable to laser capture microdissection with well-balanced RNA quality and tissue morphology.

Abstract

Assessing how gene expression analysis by RNA sequencing (RNA-Seq) correlates to a unique morphology is increasingly necessary, and laser capture microdissection (LCM) is a critical research tool for discovering the genes responsible in a region of interest (ROI). Because RNA-Seq requires high-quality RNA, a sample preparation procedure that can preserve morphology and give the required quality of RNA is essential. A PAXgene®-fixed paraffin-embedded (XFPE) block can satisfy the need for high-quality RNA, but there are few reports on adapting the method for LCM, such as how small an ROI is analyzable by RNA-Seq. In this study, we confirmed the morphology and preservation of RNA in XFPE and then assessed the relationship between the size of pieces cut by LCM and their RNA quality. In XFPE, the morphology was similar to that in alcohol-based fixed samples, the quality of the RNA extracted from a whole sample was excellent, that is equivalent to that of a fresh frozen sample, and the quality was maintained over one year later. Three sizes of pieces—large (25,000 µm2), medium (5,000 µm2), and small (1,000 µm2)—were cut by LCM so that the total areas of the sections cut per size were the same. RNA quality was found to be best preserved when tissue was cut into pieces of over 5,000 µm2. In summary, XFPE exhibits good morphology and excellent preservation of RNA quality. Furthermore, it can be a good tool when used with LCM and RNA-Seq, giving well-balanced RNA quality and tissue morphology in the ROI.