Comparing RNA extraction protocols from formalin-fixed paraffin-embedded microcore samples.

The ability to analyze intratumoral heterogeneity is of great interest for both diagnostic and basic research purposes. However, currently available dissection techniques are unsuitable for routine use and hard to access financially. Recently, a novel microcore-based dissection technique has been developed by the company Excilone for studying tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) microcore samples. The use of FFPE biological samples for transcriptomic studies, coupled with their small size, remains a real barrier to dissection applications. The efficacy of five commercially available RNA extraction kits were analyzed on microcores collected from human and mice FFPE tissues. Thirty microcore samples of healthy tissue (human uterus and stomach, and murine liver and spleen) were collected and distributed equally and randomly to the five kits assessed. Microcores were collected directly from paraffin blocks using 200 µm inner diameter needles with a sample depth, variable regarding to tissue type, ranging from 450 to 600 µm. Overall RNA yield and RNA fragmentation were evaluated, and RT-qPCR analyses were carried out after deparaffinization and compared to non-deparaffinization protocols. RNA yields and RNA fragmentation varied considerably between kits and FFPE tissues analyzed. Although the main limitation of this technique is the small initial sample size, differences in qPCR efficiency were also observed. Interestingly, no significant differences were observed between deparaffinized and non-deparaffinized microcore samples. Ultimately, we demonstrate the feasibility of using FFPE microcore samples for sensitive molecular biology applications, both with and without deparaffinization. The importance of setting up an optimized workflow was emphasized by significant differences observed in outcomes of the different protocols.