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In situ 10-cell RNA sequencing in tissue and tumor biopsy samples.

ABSTRACT: Single-cell transcriptomic methods classify new and existing cell types very effectively, but alternative approaches are needed to quantify the individual regulatory states of cells in their native tissue context. We combined the tissue preservation and single-cell resolution of laser capture with an improved preamplification procedure enabling RNA sequencing of 10 microdissected cells. This in situ 10-cell RNA sequencing (10cRNA-seq) can exploit fluorescent reporters of cell type in genetically engineered mice and is compatible with freshly cryoembedded clinical biopsies from patients. Through recombinant RNA spike-ins, we estimate dropout-free technical reliability as low as ~250 copies and a 50% detection sensitivity of ~45 copies per 10-cell reaction. By using small pools of microdissected cells, 10cRNA-seq improves technical per-cell reliability and sensitivity beyond existing approaches for single-cell RNA sequencing (scRNA-seq). Detection of low-abundance transcripts by 10cRNA-seq is comparable to random 10-cell groups of scRNA-seq data, suggesting no loss of gene recovery when cells are isolated in situ. Combined with existing approaches to deconvolve small pools of cells, 10cRNA-seq offers a reliable, unbiased, and sensitive way to measure cell-state heterogeneity in tissues and tumors.

SUBMITTER: Singh S

PROVIDER: S-EPMC6426952 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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In situ 10-cell RNA sequencing in tissue and tumor biopsy samples.

Singh Shambhavi S Wang Lixin L Schaff Dylan L DL Sutcliffe Matthew D MD Koeppel Alex F AF Kim Jungeun J Onengut-Gumuscu Suna S Park Kwon-Sik KS Zong Hui H Janes Kevin A KA

Scientific reports 20190320 1

Single-cell transcriptomic methods classify new and existing cell types very effectively, but alternative approaches are needed to quantify the individual regulatory states of cells in their native tissue context. We combined the tissue preservation and single-cell resolution of laser capture with an improved preamplification procedure enabling RNA sequencing of 10 microdissected cells. This in situ 10-cell RNA sequencing (10cRNA-seq) can exploit fluorescent reporters of cell type in genetically ...[more]

PMID: 30894605

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Project description:BackgroundThe Oncomine Dx Target Test (ODxTT) is a next-generation sequencing-based companion diagnostic test which has been recently developed; however, its analysis success rate could be improved, especially for small samples. The aim of this study was to identify the pathological factors associated with biopsy specimens that affect the analysis success rate of ODxTT.MethodsWe retrospectively investigated 119 cases subjected to ODxTT at Kanagawa Cancer Center. Data pertaining to the results of BRAF V600E mutation analysis in ODxTT and pathological factors based on microscope slides were collected. Pathological factors including tissue surface area, tumor cell count, and tumor content rate were assessed. We constructed receiver operating characteristic curves and determined the optimal cutoff values of each pathological factor. Multivariate logistic analysis was used to identify significant factors.ResultsA total of 98 of 119 samples were successfully analyzed (75.6%). The tissue surface area and tumor cell count were significantly higher in the group associated with analysis success (P < 0.001 and P = 0.011, respectively), and their optimal cutoff values were 1.04 mm2 and 375 cells, respectively. A tissue surface area > 1.04 mm2 and tumor cell count >375 cells had a positive effect on the analysis success rate of ODxTT (odds ratio [OR] 0.10; 95% confidence interval [CI]: 0.03-0.35; P < 0.001 and OR 0.25; 95% CI: 0.07-0.90; P = 0.033, respectively).ConclusionsSelecting samples with a tissue surface area > 1.04 mm2 and a tumor cell count >375 cells might improve the analysis success rate of ODxTT.Key pointsSignificant findings of the study: We found that a tissue surface area > 1.04 mm2 and tumor cell count >375 cells had a positive effect on the analysis success rate of ODxTT in the analysis of biopsy tissue samples.What this study addsIt is sometimes necessary to assess genetic alterations with a small biopsy sample in daily practice. The criteria mentioned above will help to determine which tests should be performed, ODxTT or multiple single-gene testing.

Dataset Information

In situ 10-cell RNA sequencing in tissue and tumor biopsy samples.

Publications

In situ 10-cell RNA sequencing in tissue and tumor biopsy samples.

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