Selection and testing of reference genes for accurate RT-qPCR in rice seedlings under iron toxicity.

Fabiane Igansi De Castro Dos Santos,Bianca Silva Fernandes Hoffman,Marcio Alves-Ferreira,Naciele Marini,Antonio Costa De Oliveira,Railson Schreinert Dos Santos,Niranjan Baisakh

doi:10.1371/journal.pone.0193418

Fabiane Igansi De Castro Dos Santos, Bianca Silva Fernandes Hoffman + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0193418

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Abstract

Reverse Transcription quantitative PCR (RT-qPCR) is a technique for gene expression profiling with high sensibility and reproducibility. However, to obtain accurate results, it depends on data normalization by using endogenous reference genes whose expression is constitutive or invariable. Although the technique is widely used in plant stress analyzes, the stability of reference genes for iron toxicity in rice (Oryza sativa L.) has not been thoroughly investigated. Here, we tested a set of candidate reference genes for use in rice under this stressful condition. The test was performed using four distinct methods: NormFinder, BestKeeper, geNorm and the comparative ΔCt. To achieve reproducible and reliable results, Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed. Valid reference genes were found for shoot (P2, OsGAPDH and OsNABP), root (OsEF-1a, P8 and OsGAPDH) and root+shoot (OsNABP, OsGAPDH and P8) enabling us to perform further reliable studies for iron toxicity in both indica and japonica subspecies. The importance of the study of other than the traditional endogenous genes for use as normalizers is also shown here.

Highlights

The reverse transcription quantitative PCR (RT-qPCR) has revolutionized the field of gene expression analysis
In this study we have investigated the transcriptional stability of housekeeping genes frequently used in rice (OsGAPDH, OsNABP, OsEF-1a) in distinct plant organs and genotypes, comparing their expression in conditions of iron stress against control conditions
We tested three traditional reference genes used in many previous studies (OsGAPDH, OsNABP and OsEF-1a) and nine novel candidate reference genes selected from the Genevestigator database (Table 1)

Summary

Introduction

The reverse transcription quantitative PCR (RT-qPCR) has revolutionized the field of gene expression analysis. Being one of the most sensitive, precise and reproducible techniques to quantify specific RNAs, it is employed in diverse fields and became the most common method for validating microarray and RNAseq data [1,2,3]. It is an extremely powerful technique, RT-qPCR suffers from certain pitfalls, and the normalization with a housekeeping gene, which expression remains constant between cells of different tissues and under different experimental conditions, is an important step of this analysis [4].

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