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

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.

Arachis hypogaea gene expression atlas for fastigiata subspecies of cultivated groundnut to accelerate functional and translational genomics applications

“…This study also provides the stable transcripts with the least variability among tissues involved in basic metabolic processes. It is confirmed from many previous studies that it is very crucial to use reference genes specific to the trait under study and various plant developmental stages (Reddy et al, 2013;dos Santos et al, 2018;Sinha et al, 2015). Such information on tissue-specific as well as constitutively expressed genes is not available for groundnut research community.…”

Gene Expression of Transient Receptor Potential Channels in Peripheral Blood Mononuclear Cells of Inflammatory Bowel Disease Patients

“…Data representing the relative expression of detected mRNA normalized to GAPDH mRNA was used as a calibrator for comparative analysis. RT-qPCR was performed in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [ 19 , 20 ]. The relative expression data was calculated according to the 2 -ΔΔCt method.…”

Evaluation of reference genes and expression of key genes involved in the isoprenoid metabolic pathway of rice leaves after infection by the Southern rice black-streaked dwarf virus

“…EF1α has been identified as a stable reference gene in rice subject to low temperatures [36], and UBQ10 has been recommended as a reliable reference gene in rice plant leaves subject to salt stress [28]. GAPDH is commonly regarded as the "classical" reference gene [45], and has been suggested as a reliable reference gene in rice seedlings suffering from iron toxicity [35]. However, we found the expression of GAPDH to be quite variable.…”

“…There is, however, no universal reference gene suitable for all species or experimental conditions [34]. Although many studies have assessed reference genes in different tissues and development stages of rice plants, and after subjecting plants to different kinds of stress [28,[35][36][37][38][39][40][41], to the best of our knowledge, no one has attempted to identify stable reference genes in the leaves of SRBSDVinfected rice plants.…”