Validation of Potential Reference Genes for qPCR in Maize across Abiotic Stresses, Hormone Treatments, and Tissue Types

Yueai Lin,Guangtang Pan,Shibin Gao,Hailan Liu,Suzhi Zhang,Moju Cao,Hai Lan,Chenlu Zhang,Jian Liu,Tingzhao Rong,Jitesh Pratap

doi:10.1371/journal.pone.0095445

Abstract

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α), tubulin beta (β-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types.

Highlights

A determination of the gene expression pattern is very important for the functional exploration of a target gene
Despite the rapid exploration of the maize genome and the growing requirement for the deep biological understanding of gene function aided by gene expression patterns, very limited information is available on the expression stability of reference genes in maize under particular experimental conditions
We evaluated the expression stability of ten potential reference genes, eight of which (GAPDH, elongation factor 1a (EF1a), ACT2, b-TUB, UBQ9, CYP, eukaryotic initiation factor 4A (EIF4A), and UBQ7) were commonly used internal control genes and two of which (GLU1 and GRP) were novel candidate reference genes identified from our microarray datasets of salt and abscisic acid (ABA) treatments, respectively, in a set of 26 maize samples collected from different experimental conditions with respect to abiotic stresses, hormones and tissue types

Summary

Introduction

A determination of the gene expression pattern is very important for the functional exploration of a target gene. The commonly used reference genes, such as glyceraldehyde-3phosphate dehydrogenase (GAPDH), elongation factor 1a (EF1a), tubulin b-chain (b-TUB), polyubiquitin (UBQ), 18S ribosomal RNA (18S rRNA), and b-actin (ACT), should be carefully handled as internal controls, because their transcript levels are variable under particular experimental conditions [2,3,7,9,10,11,12]. These existing reference genes are limited to particular experimental conditions and designs. It is necessary to evaluate potential reference genes under different experimental conditions prior to their use in RT-qPCR data normalization

Methods

Results

Conclusion