Dataset Information

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress.

ABSTRACT: Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI, dnaG, and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytvI, dnaG, and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.

SUBMITTER: Zhao N

PROVIDER: S-EPMC8430261 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress.

Zhao Ning N Xu Junnan J Jiao Lingxia L Qiu Mengzhen M Zhang Jie J Wei Xinyuan X Fan Mingtao M

Frontiers in microbiology 20210827

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate R ...[more]

PMID: 34512609

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Project description:Compared with the traditional gene expression techniques of quantitative analysis, RT-qPCR is most widely used because of its low cost, time-saving, and accuracy. It is essential to introduce suitable internal reference genes as reference corrections in RT-qPCR experiments to reduce the RNA quality and reverse transcription efficiency of different samples. In our study, we chose the candidate internal reference genes of alfalfa from transcriptome sequence datasets (162 RNA-seq sequencing data) through comparative analysis. Finally, 10 candidate reference genes were selected. These candidate reference gene expressions were determined by RT-qPCR under five abiotic stresses of drought, alkali, high temperature, low temperature, and acid treatments. The stability index of these candidate genes was calculated and evaluated correspondently using specific software and different lgorithms, such as GeNorm, Normfinder, Bestkeeper, ΔCt method, and an online analysis tool RefFinder. Then the appropriate candidate genes were screened strictly; and validated by the phyA gene. GAPDH and Actin are taken as traditional reference genes on gene expression of quantitative analysis commonly used in alfalfa, Our results showed GAPDH and Actin aren't the most appropriate reference genes of alfalfa under different abiotic stresses, under alkaline stress, the optimal reference gene is UBL-2a, and the optimal combination of reference genes is MS.65,463 (some candidate reference genes haven't been annotated yet, using gene ID abbreviation number of Medicago sativa L. instead )and UBL-2a; Under drought stress, the optimal reference gene is Ms.33,066, and the optimal combination of reference genes is MS.65,463 and UBL-2a; Under high-temperature stress, the optimal reference gene is Actin, and the optimal combination of reference genes is Rer1, Actin, MS.00617, MS.74,923, UBL-2a, MS.33,066, MS.99,505, and MS.65,463; Under low-temperature stress, the optimal reference gene is Actin, and the optimal combination of reference genes is Rer1, Actin, MS.99,505, MS.073307, and UBL-2a. The optimal reference genes and their combinations need further validation under acid stress. This paper provides scientific evidence for quantitative analysis of the genes of alfalfa.

Dataset Information

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress.

Publications

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of <i>Alicyclobacillus acidoterrestris</i> Under Acid Stress.

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