The choice of negative control antisense oligonucleotides dramatically impacts downstream analysis depending on the cellular background

Luca Ducoli,Jay W Shin,Takeya Kasukawa,Harukazu Suzuki,Eliane Sibler,Jordan A Ramilowski,Imad Abugessaisa,Chung-Chau Hon,Michihira Tagami,Michiel J L De Hoon,Masayoshi Itoh,Piero Carninci,Akira Hasegawa,Naoto Kondo,Michael Detmar,Saumya Agrawal

doi:10.1186/s12863-021-00992-1

Abstract

BackgroundThe lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism’s physiological function. Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. The recent technological boost in sequencing approaches unveiled long noncoding RNAs (lncRNAs) as prominent regulatory players of various gene expression levels in a cell-type-specific manner.ResultsTo investigate the potential roles of lncRNAs in vascular biology, we performed antisense oligonucleotide (ASO) knockdowns of lncRNA candidates specifically expressed either in human lymphatic or blood vascular endothelial cells (LECs or BECs) followed by Cap Analysis of Gene Expression (CAGE-Seq). Here, we describe the quality control steps adopted in our analysis pipeline before determining the knockdown effects of three ASOs per lncRNA target on the LEC or BEC transcriptomes. In this regard, we especially observed that the choice of negative control ASOs can dramatically impact the conclusions drawn from the analysis depending on the cellular background.ConclusionIn conclusion, the comparison of negative control ASO effects on the targeted cell type transcriptomes highlights the essential need to select a proper control set of multiple negative control ASO based on the investigated cell types.

Highlights

The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism’s physiological function
Lymphatic endothelial cell (LEC) and Blood vascular endothelial cell (BEC) were first transfected in duplicates with eight antisense oligonucleotide (ASO) independently
After mapping and Cap analysis of gene expression (CAGE) promoter quantification, the impacts of negative control ASOs on LECs and BECs were evaluated by performing Differential Expression (DE) and Gene Ontology (GO) analysis and in vitro cellular assays (Fig. 1b)

Summary

Introduction

The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism’s physiological function Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. In the cytoplasm, lncRNAs can function as a scaffold for protein complexes regulating mRNA stability, translation, and decay [11,12,13,14]. This vast functional repertoire of lncRNAs has led to the novel idea of RNA as a central molecule in the regulation of gene functions. Mutation and/or overexpression of lncRNAs have been implicated in a multitude of human diseases, proposing lncRNA signatures as possible diagnostic factors of malignant conditions [17]

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Discussion

Conclusion