The Selection and Validation of Reference Genes for mRNA and microRNA Expression Studies in Human Liver Slices Using RT-qPCR.

Tomáš Zárybnický,Petra Matoušková,Martin Ambrož,Iva Boušová,Zdeněk Šubrt,Lenka Skálová

doi:10.3390/genes10100763

Tomáš Zárybnický, Petra Matoušková + Show 4 more

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https://doi.org/10.3390/genes10100763

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Journal: Genes	Publication Date: Sep 28, 2019
Citations: 10	License type: CC BY 4.0

Affiliation: Charles University, University Hospital Kralovske Vinohrady

Abstract

The selection of a suitable combination of reference genes (RGs) for data normalization is a crucial step for obtaining reliable and reproducible results from transcriptional response analysis using a reverse transcription-quantitative polymerase chain reaction. This is especially so if a three-dimensional multicellular model prepared from liver tissues originating from biologically diverse human individuals is used. The mRNA and miRNA RGs stability were studied in thirty-five human liver tissue samples and twelve precision-cut human liver slices (PCLS) treated for 24 h with dimethyl sulfoxide (controls) and PCLS treated with β-naphthoflavone (10 µM) or rifampicin (10 µM) as cytochrome P450 (CYP) inducers. Validation of RGs was performed by an expression analysis of CYP3A4 and CYP1A2 on rifampicin and β-naphthoflavone induction, respectively. Regarding mRNA, the best combination of RGs for the controls was YWHAZ and B2M, while YWHAZ and ACTB were selected for the liver samples and treated PCLS. Stability of all candidate miRNA RGs was comparable or better than that of generally used short non-coding RNA U6. The best combination for the control PCLS was miR-16-5p and miR-152-3p, in contrast to the miR-16-5b and miR-23b-3p selected for the treated PCLS. Our results showed that the candidate RGs were rather stable, especially for miRNA in human PCLS.

Highlights

A set of potential reference genes (RGs) for mRNA was tested in thirty-five human liver samples using
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), with the combination of YWHAZ/ACTB identified in geNorm and RefFinder as the most suitable for gene normalization
Two sets of potential RGs for mRNA and miRNA were tested in human precision-cut human liver slices (PCLS) at multiple time points during a 24 h incubation using RT-qPCR

Summary

Introduction

Choosing suitable reference genes (RGs) for the normalization of gene expression is always a dilemma. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a very potent tool with high sensitivity, accuracy, specificity, reproducibility, and relatively low cost in comparison to microarrays or high-throughput sequencing [13]. There have been no studies to properly validate the stability of RGs despite the spreading usage of PCLS, PCLS are a useful model to study liver injury as. A number of prerequisites are involved in searching for suitable and effective RGs to elicit reproducible results from any RT-qPCR analysis including a stable expression among the analyzed samples as well as preventing the samples from being affected by the experimental conditions [13,16,17]

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