Correction: Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Soybean Tissues under Various Abiotic Stress Conditions

Abstract

Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid) treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue-and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/ 60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used.

Genome-Wide Analysis of the WOX Gene Family and Function Exploration of GmWOX18 in Soybean

“…qRT-PCR was carried out using the TransScript tip green qPCR SuperMix kit, following the manufacturer’s instructions (Transgen). The Glyma.19G147900.1 gene was used as an internal control [55]. Statistical analyses were performed using a t-test, and p < 0.05 and < 0.01 were considered significant and extremely significant differences, respectively.…”

Selection and Validation of Reference Genes for the qRT-PCR Assays of Populus ussuriensis Gene Expression under Abiotic Stresses and Related ABA Treatment

“…Under these stresses, increased levels of endogenous ABA can upregulate many stress-related genes in an ABA-dependent manner [37]. Numerous studies have identified and validated RGs in rice [38], maize [39], soybean [40,41], and carrot [42] under abiotic stress. However, there are currently no reports of RGs that are universally and stably expressed under abiotic stress in P. ussuriensis.…”

“…For example, 60S rRNA was stably expressed among different developmental stages in Panax ginseng [50]. Others include RPL30, which has been used as an RG in roots and shoots of soybean seedlings under cold stress and ABA treatment [40], and RPL2, which is stably expressed under nitrogen, cold, and light stress in tomato [8]. Ribosomal protein genes are also widely used as RGs in animals [51][52][53].…”

The Drought-Mediated Soybean GmNAC085 Functions as a Positive Regulator of Plant Response to Salinity

“…Preparation for reactions, thermal profile and melting curve analysis in RT-qPCR assay (Mastercycler®ep realplex, Eppendorf, Hamburg, Germany) was described in our previous study [97]. Fbox [107] was used as the reference gene for normalization based on the 2 −∆Ct method [108]. LinRegPCR software (version 2020.2, Academic Medical Center, Amsterdam, The Netherlands) was used to calculate the efficiency of PCR reactions.…”