Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting

Fabian Link,Julia Schumann,Knut Krohn

doi:10.1038/s41598-019-49241-7

Fabian Link, Julia Schumann + Show 1 more

Open Access

https://doi.org/10.1038/s41598-019-49241-7

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Abstract

Reliable quantification of miRNA expression by qRT-PCR crucially depends on validated housekeepers for data normalization. Here we present thoroughly tested miRNAs eligible as references in immunological studies utilizing endothelial cells and macrophages, respectively. Endothelial cells (cell line: TIME) and macrophages (cell line: RAW264.7) were treated with various pro- and anti-inflammatory mediators (cytokines, LPS, unsaturated fatty acids) given as either single substances or in combination. Isolated RNA was screened for stably expressed miRNAs by next generation sequencing. Housekeeper candidates were thereafter validated by means of two independent quantification techniques: qRT-PCR for relative quantification and ddPCR for absolute quantification. Both methods consistently confirmed the suitability of let-7g-5p, let-7i-5p, miR-127-3p and miR-151a-5p in cytokine/fatty acid-treated TIME and miR-16-5p, miR-27b-3p, miR-103a-3p and miR-423-3p in LPS/fatty acid-treated RAW264.7, respectively as housekeeping miRNAs. With respect to abundancy and over all expression stability the miRNAs miR-151a-5p (cell line: TIME) as well as miR-27b-3p and miR-103a-3p (cell line: RAW264.7) can be particularly recommended for normalization of qRT-PCR data.

Highlights

Reliable quantification of miRNA expression by quantification is the Real-Time polymerase chain reaction (qRT-PCR) crucially depends on validated housekeepers for data normalization
Housekeeping genes typically used for messenger RNA quantification like GAPDH and β-actin are considerably larger than miRNAs
In TIME treated with the cytokines IL-1β, TNF-α and IFN-γ (5 ng/ml each) 1173 different miRNAs were detected compared with 1093 miRNAs in unstimulated control cells

Summary

Introduction

Reliable quantification of miRNA expression by qRT-PCR crucially depends on validated housekeepers for data normalization. Housekeeper candidates were thereafter validated by means of two independent quantification techniques: qRT-PCR for relative quantification and ddPCR for absolute quantification Both methods consistently confirmed the suitability of let-7g-5p, let-7i-5p, miR-127-3p and miR-151a-5p in cytokine/ fatty acid-treated TIME and miR-16-5p, miR-27b-3p, miR-103a-3p and miR-423-3p in LPS/fatty acidtreated RAW264.7, respectively as housekeeping miRNAs. With respect to abundancy and over all expression stability the miRNAs miR-151a-5p (cell line: TIME) as well as miR-27b-3p and miR-103a-3p (cell line: RAW264.7) can be recommended for normalization of qRT-PCR data. There is evidence that the expression rate of these housekeepers differs among tissue types and shows inter-individual variations[22] These normalizers seem to be affected by the surrounding milieu, e.g. the presence of cytokines[23,24,25]. There is a need for the identification of stably expressed housekeeper miRNAs with respect to the experimental setting

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