An optimised protocol harnessing laser capture microdissection for transcriptomic analysis on matched primary and metastatic colorectal tumours to identify novel biomarker

Purpose: To develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations Methods: Snap frozen biopsies of matched primary tumour, metastasis and the associated normal tissues were microdissected to obtain stromal and epithelial components. RNA was extracted from the respective microdissected samples and subsequently subjected to RNA sequencing. Results: We demonstrated an optimised LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). This framework helped to uncover multiple differentially expressed cancer-associated genes and qPCR verification of 9 chosen targets confirmed the high fidelity of the RNA sequencing process. Conclusion: This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution. Snap frozen biopsies of matched primary tumour, metastasis and the associated normal tissues were microdissected to obtain stromal and epithelial components. RNA was extracted from the respective microdissected samples and subsequently subjected to RNA sequencing.