An Optimised Protocol Harnessing Laser Capture Microdissection for Transcriptomic Analysis on Matched Primary and Metastatic Colorectal Tumours

Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoredu...

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Bibliographic Details
Published in:Scientific reports 2020-01, Vol.10 (1), p.682-682, Article 682
Main Authors: Ong, Chin-Ann J, Tan, Qiu Xuan, Lim, Hui Jun, Shannon, Nicholas B, Lim, Weng Khong, Hendrikson, Josephine, Ng, Wai Har, Tan, Joey W S, Koh, Kelvin K N, Wasudevan, Seettha D, Ng, Cedric C Y, Rajasegaran, Vikneswari, Lim, Tony Kiat Hon, Ong, Choon Kiat, Kon, Oi Lian, Teh, Bin Tean, Tan, Grace H C, Chia, Claramae Shulyn, Soo, Khee Chee, Teo, Melissa C C
Format: Article
Language:English
Subjects:
Colorectal cancer
Colorectal Neoplasms - genetics
Colorectal Neoplasms - secondary
Colorectal Neoplasms - surgery
DNA-directed RNA polymerase
Female
Gene expression
Gene Expression Profiling - methods
Gene Expression Regulation, Neoplastic
Genomic analysis
High-Throughput Nucleotide Sequencing
Humans
Krukenberg Tumor - genetics
Krukenberg Tumor - surgery
Laser Capture Microdissection - methods
Metastases
Next-generation sequencing
Ovarian Neoplasms - genetics
Ovarian Neoplasms - surgery
Polymerase chain reaction
Ribonucleic acid
RNA
Sequence Analysis, RNA
Specimen Handling
Surgery
Tumors
Workflow
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Summary:Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework's potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. 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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-019-55146-2