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

PloS One
Fabiane Igansi de Castro Dos SantosAntonio Costa de Oliveira

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.

References

Jan 5, 2000·Journal of Biotechnology·O ThellinE Heinen
Aug 19, 2000·BioTechniques·T SuzukiD R Crawford
Feb 16, 2002·Methods : a Companion to Methods in Enzymology·K J Livak, T D Schmittgen
Apr 9, 2005·Genes and Immunity·J HuggettA Zumla
Sep 29, 2005·Journal of Experimental Botany·Nathalie NicotDanièle Evers
Oct 26, 2005·Journal of Computational Biology : a Journal of Computational Molecular Cell Biology·Sheng Zhao, Russell D Fernald
May 13, 2006·Biochemical and Biophysical Research Communications·Mukesh JainJitendra P Khurana
Jan 29, 2008·BMC Genomics·Dong WangChengchao Zheng
Apr 7, 2010·PLoS Pathogens·Laura CamarenaMichael Snyder
Aug 20, 2011·Plant & Cell Physiology·Bas J W DekkersLeónie Bentsink
Feb 1, 2012·Plant Molecular Biology·Fuliang XieBaohong Zhang
May 5, 2012·Wiley Interdisciplinary Reviews. RNA·Arjun N SasikumarTerri Goss Kinzy
Aug 1, 2015·Rice·Takanori KobayashiNaoko K Nishizawa
Nov 18, 2016·Scientific Reports·Rasmus RydbirkTomasz Brudek
Mar 18, 2017·Genetics and Molecular Biology·Railson Schreinert Dos SantosAntonio Costa de Oliveira

❮ Previous
Next ❯

Methods Mentioned

BETA
reverse
RNAseq
electrophoresis
PCR

Software Mentioned

geNORM
BestKeeper
Finder
time PCR Miner
NormFinder
Genevestigator
RefFinder
Norm
RefGenes
Real

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.