ABSTRACT

Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression.

We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-βH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples.

We identified a total of 264 genes stably expressed in EndoC-βH1 cells and human islets following cytokines – or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.

摘要

人类胰腺 β 细胞暴露于促炎细胞因子或代谢应激源分别用于模拟与 1 型和 2 型糖尿病相关的事件。定量实时 PCR 通常用于量化基因表达的变化。为基因表达标准化选择最合适的参考基因是获得准确和可靠结果的重要先决条件。没有普遍适用的参考基因，常用参考基因​​的人类β细胞表达可以被不同的压力源改变。在这里，我们旨在鉴定人类 β 细胞中最稳定表达的基因，以使定量实时 PCR 基因表达正常化。

我们使用来自人类胰腺β细胞系 EndoC-βH1、暴露于细胞因子或游离脂肪酸棕榈酸酯的人类胰岛的综合 RNA 测序数据，以确定最稳定表达的基因。根据基因的显着性水平（调整后的P值 > 0.05）、倍数变化（|倍数变化| <1.5）和变异系数 <10% 过滤基因。候选参考基因通过独立样本中的定​​量实时 PCR 进行验证。

我们确定了总共 264 个基因在 EndoC-βH1 细胞和人类胰岛细胞因子或棕榈酸盐诱导的压力下稳定表达，显示出低变异系数。前五个基因ARF1、CWC15、RAB7A、SIAH1和VAPA的定量实时 PCR 验证证实了它们在大多数测试条件下的表达稳定性。在独立样本中的进一步验证表明，ACTB和VAPA表达的几何平均值可用作人类 β 细胞中可靠的标准化因子。