Identification of the best housekeeping gene for RT-qPCR analysis of human pancreatic organoids.

Alessandro Cherubini,Francesco Rusconi,Lorenza Lazzari,Zoltán Rakonczay

doi:10.1371/journal.pone.0260902

Alessandro Cherubini, Francesco Rusconi + Show 2 more

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https://doi.org/10.1371/journal.pone.0260902

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Abstract

In the last few years, there has been a considerable increase in the use of organoids, which is a new three-dimensional culture technology applied in scientific research. The main reasons for their extensive use are their plasticity and multiple applications, including in regenerative medicine and the screening of new drugs. The aim of this study was to better understand these structures by focusing on the choice of the best housekeeping gene (HKG) to perform accurate molecular analysis on such a heterogeneous system. This feature should not be underestimated because the inappropriate use of a HKG can lead to misleading data and incorrect results, especially when the subject of the study is innovative and not totally explored like organoids. We focused our attention on the newly described human pancreatic organoids (hPOs) and compared 12 well-known HKGs (ACTB, B2M, EF1α, GAPDH, GUSB, HPRT, PPIA, RNA18S, RPL13A TBP, UBC and YWHAZ). Four different statistical algorithms (NormFinder, geNorm, BestKeeper and ΔCt) were applied to estimate the expression stability of each HKG, and RefFinder was used to identify the most suitable genes for RT-qPCR data normalization. Our results showed that the intragroup and intergroup comparisons could influence the best choice of the HKG, making clear that the identification of a stable reference gene for accurate and reproducible RT-qPCR data normalization remains a critical issue. In summary, this is the first report on HKGs in human organoids, and this work provides a strong basis to pave the way for further gene analysis in hPOs.

Highlights

In the last few years, there has been a remarkable increase in the use of organoid technology in scientific research
The curves showed that the RT-qPCR efficiency of each primer pair ranged between 95% and 105% (S2 Table), which is in agreement with MIQE guidelines [11]
We present the first study on human pancreatic organoids (hPOs) focusing on the identification and validation of a suitable set of housekeeping gene (HKG) to use in gene expression analysis during short- and long-term culture

Summary

Introduction

In the last few years, there has been a remarkable increase in the use of organoid technology in scientific research. Organoids are three-dimensional cellular bodies that can be generated from pluripotent stem cells (embryonic or induced) or adult tissue-specific ancestor cells [1]. The main reasons for their considerable use are their plasticity and multiple applications, including as new models to study specific diseases [2], in regenerative medicine [3] and the screening of new drugs [4]. Correct choice of housekeeping gene for human pancreatic organoids endocrine cells derived from human pancreas organoids for the cell-based therapy of Type 1 diabetes” project number 668350. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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