Dataset Information

Identification of suitable reference genes for quantitative reverse transcription PCR in Luffa (Luffa cylindrica).

ABSTRACT: Reverse transcription real-time quantitative PCR is widely used to quantify gene expression. Reference genes are usually used as internal controls to measure the target gene expression level. To date, there is no consensus on the use of systematically validated reference genes in different tissues of Luffa. This study evaluated the expression stability of 11 candidate reference genes in different tissues using five algorithms (BestKeeper, comparative delta-Ct method, GeNorm, NormFinder, and RefFinder). Protein phosphatase 2A was the most stable gene, while alpha Tubulin was the least stable. The relative expression of ethylene-related genes in different tissues was also analyzed to reveal their role in sex determination. This study provides the basis for using suitable reference genes to evaluate targeted gene expression.

Supplementary information

The online version contains supplementary material available at 10.1007/s12298-022-01182-8.

SUBMITTER: Zhao G

PROVIDER: S-EPMC9110621 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Identification of suitable reference genes for quantitative reverse transcription PCR in Luffa (Luffa cylindrica).

Zhao Gangjun G Wang Meng M Gan Yaqin Y Gong Hao H Li Junxing J Zheng Xiaoming X Liu Xiaoxi X Zhao Siying S Luo Jianning J Wu Haibin H

Physiology and molecular biology of plants : an international journal of functional plant biology 20220401 4

Reverse transcription real-time quantitative PCR is widely used to quantify gene expression. Reference genes are usually used as internal controls to measure the target gene expression level. To date, there is no consensus on the use of systematically validated reference genes in different tissues of Luffa. This study evaluated the expression stability of 11 candidate reference genes in different tissues using five algorithms (BestKeeper, comparative delta-Ct method, GeNorm, NormFinder, a ...[more]

PMID: 35592479

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Project description:Gene expression analysis largely improves our understanding of the molecular basis underpinning various plant biological processes. Stable reference genes play a foundational role during the normalization of gene expression levels. However, until now, there have been few reference genes suitable for ginger reverse transcription-quantitative PCR (RT-qPCR) research. In this study, 29 candidate reference genes with stable expression patterns across multiple ginger tissues and 13 commonly used reference genes were selected to design RT-qPCR primers. After amplification specificity validation, 32 candidates were selected and further evaluated by RT-qPCR using samples from various organs subjected to NaCl, drought, heat, waterlogging, and chilling stress. Four strategies, including delta-CT, BestKeeper, geNorm, and NormFinder, were used to rank the stability of reference genes, and the ranks produced by these four strategies were comprehensively evaluated by RefFinder to determine the final rank. Overall, the top three stability reference genes indicated by RefFinder were RBP > ATPase > 40S_S3. Their expression pattern correlation analysis showed that the coefficients among each pair of RBP, ATPase, and 40S_S3 were larger than 0.96, revealing consistent and stable expression patterns under various treatments. Then, the expression of three pathogenesis-related (PR) genes and seven MYB genes in rhizomes during postharvest storage and subjected to pathogen infection was normalized by RBP, ATPase, 40S_S3, RBP and ATPase, ATPase and 40S-S3, and RBP and 40S-S3. The results showed that PR and MYB genes were induced by postharvest deterioration and pathogen infection. The correlation coefficients of RBP/ATPase, RBP/40S_S3, ATPase/40S_S3, RBP and ATPase/ATPase and 40S-S3, RBP and ATPase/RBP and 40S-S3, and ATPase and 40S-S3/RBP and 40S-S3 were 0.99, 0.96, 0.99, 0.99, 1.00, and 1.00, respectively, which confirmed the stability of these three reference genes in postharvest biology studies of ginger. In summary, this study identified appropriate reference genes for RT-qPCR in ginger and facilitated gene expression studies under biotic and abiotic stress conditions.

Dataset Information

Identification of suitable reference genes for quantitative reverse transcription PCR in Luffa (Luffa cylindrica).

Supplementary information

Publications

Identification of suitable reference genes for quantitative reverse transcription PCR in <i>Luffa</i> (<i>Luffa cylindrica</i>).

Similar Datasets

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