Stability analysis of reference genes for RT-qPCR assays involving compatible and incompatible Ralstonia solanacearum-tomato ‘Hawaii 7996’ interactions

Albuquerque, Greecy Mirian Rodrigues; Fonseca, Fernando; Boiteux, L. S.; Borges, R. C. F.; Miller, Robert N.; Lopes, C. A.; Souza, Elineide Barbosa de; Fonseca, M. E. N.

doi:10.1038/s41598-021-97854-8

Cited by 11 publications

(7 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There have been several reports assessing RGs stability in plant-pathogen systems involving compatible- and incompatible interactions. Albuquerque et al 32 studied RGs expression stability in tomato during interaction with Ralstonia solanacearum isolates displaying contrasting virulence profiles. Interestingly, identified most stable RGs differed between compatible and incompatible interaction analysis groups ( PDS + ACT vs TIP41 + EF1A , respectively).…”

Section: Discussionmentioning

confidence: 99%

Reference genes expression stability in Avena sativa L. during compatible and incompatible interactions with Puccinia graminis

Sowa

Sozoniuk

Toporowska

et al. 2022

Sci Rep

View full text Add to dashboard Cite

A reliable qPCR experiment requires the selection of reference genes with a stable level of expression in a given experimental system. This study attempts to determine the reference genes (RGs) for the A. sativa–P. graminis experimental setup. We evaluated nine candidate reference genes in A. sativa (oat line Pg4 and the cultivar Kasztan) during compatible and incompatible interactions with different pathotypes of Puccinia graminis f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). We found that the most appropriate combination of RGs for RT-qPCR data normalization were HNR (heterogeneous nuclear ribonucleoprotein 27C) + EF1A (elongation factor 1-alpha) + EIF4A (eukaryotic initiation factor 4A-3). The worst candidates for normalization in this dataset were CYP (cyclophilin) and TUA (alpha tubulin). Identified reference genes are suitable candidates for the standardization of gene expression studies in the A. sativa–P. graminis interaction system and potentially other related pathogens. To date, this is the first report of RGs selection in this pathosystem.

show abstract

Section: Discussionmentioning

confidence: 99%

Reference genes expression stability in Avena sativa L. during compatible and incompatible interactions with Puccinia graminis

Sowa

Sozoniuk

Toporowska

et al. 2022

Sci Rep

View full text Add to dashboard Cite

show abstract

“…The stability of gene expression level was analyzed using the geNorm (14,15), NormFinder (16,17), and BestKeeper software (18,19).…”

Section: Expression Stability Analysismentioning

confidence: 99%

“…The data analysis was first performed using geNorm (14). The Ct value of 25 samples was converted into a relative quantitative Q-value using the following formula: Q = 2-Ct ( Ct=Ct sample-Ct min).…”

Section: Genormmentioning

confidence: 99%

Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae

Zhou

Yuan

et al. 2022

Front. Vet. Sci.

View full text Add to dashboard Cite

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), which resulting in considerable economic losses in pig farming globally. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a major tool for gene expression studies. However, no internal reference genes for normalization of RT-qPCR data of M. hyopneumoniae have been reported. The aim of this study was to screen the most stable genes for RT-qPCR analysis in M. hyopneumoniae under different conditions. Therefore, a total of 13 candidate internal reference genes (rpoC, Lipo, sgaB, oppB, hypo621, oppF, gyrB, uvrA, P146, prfA, proS, gatB, and hypo499) of M. hyopneumoniae filtered according to the reported quantitative proteomic analysis and the 16S rRNA internal reference gene frequently used in other bacteria were selected for RT-qPCR analysis. The mRNAs from different virulence strains (168, 168 L, J, NJ, and LH) at five different growth phases were extracted. The corresponding cycle threshold (Ct) values of the 25 reverse transcribed cDNAs using the 14 candidate genes were determined. Different internal reference genes or combinations were then screened for expression stability analysis using various statistical tools and algorithms, including geNorm, BestKeeper, and NormFinder software, to ensure the reliability of the analysis. Through further comprehensive evaluation of the RefFinder software, it is concluded that the gatB gene was the most suitable internal reference gene for samples of the different virulence strains in different growth phases for M. hyopneumoniae, followed by prfA, hypo499, and gyrB.

show abstract

“…and oligos for Tip41, actin and ubiquitin of L. styraciflua were evaluated as reference genes (all primers are listed in Table S1). The geometric mean [51] between Tip41 and actin was used as a references [52,53].…”

Section: Rna Extraction and Gene Expressionmentioning

confidence: 99%

Insights of the Neofusicoccum parvum–Liquidambar styraciflua Interaction and Identification of New Cysteine-Rich Proteins in Both Species

Vázquez-Avendaño

Rodríguez-Haas

Velázquez-Delgado

et al. 2021

JoF

View full text Add to dashboard Cite

Neofusicoccum parvum belongs to the Botryosphaeriaceae family, which contains endophytes and pathogens of woody plants. In this study, we isolated 11 strains from diseased tissue of Liquidambar styraciflua. Testing with Koch’s postulates—followed by a molecular approach—revealed that N. parvum was the most pathogenic strain. We established an in vitro pathosystem (L. styraciflua foliar tissue–N. parvum) in order to characterize the infection process during the first 16 days. New CysRPs were identified for both organisms using public transcriptomic and genomic databases, while mRNA expression of CysRPs was analyzed by RT-qPCR. The results showed that N. parvum caused disease symptoms after 24 h that intensified over time. Through in silico analysis, 5 CysRPs were identified for each organism, revealing that all of the proteins are potentially secreted and novel, including two of N. parvum proteins containing the CFEM domain. Interestingly, the levels of the CysRPs mRNAs change during the interaction. This study reports N. parvum as a pathogen of L. styraciflua for the first time and highlights the potential involvement of CysRPs in both organisms during this interaction.

show abstract

Stability analysis of reference genes for RT-qPCR assays involving compatible and incompatible Ralstonia solanacearum-tomato ‘Hawaii 7996’ interactions

Cited by 11 publications

References 41 publications

Reference genes expression stability in Avena sativa L. during compatible and incompatible interactions with Puccinia graminis

Reference genes expression stability in Avena sativa L. during compatible and incompatible interactions with Puccinia graminis

Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae

Insights of the Neofusicoccum parvum–Liquidambar styraciflua Interaction and Identification of New Cysteine-Rich Proteins in Both Species

Contact Info

Product

Resources

About