Optimization of reference genes for qRT-PCR analysis of microRNA expression under abiotic stress conditions in sweetpotato

The aim of this study was to select the most suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) of spotted sea bass (Lateolabrax maculatus), an important commercial marine fish in Pacific Asia, under normal physiological and salinity stress conditions. A total of 9 candidate reference genes (HPRT, GAPDH, EF1A, TUBA, RPL7, RNAPol II, B2M, ACTB and 18S rRNA) were analyzed by qRT-PCR in 10 tissues (intestine, muscle, stomach, brain, heart, liver, gill, kidney, pectoral fins and spleen) of L. maculatus. Four algorithms, geNorm, NormFinder, BestKeeper, and comparative ΔCt method, were used to evaluate the expression stability of the candidate reference genes. The results showed the 18S rRNA was most stable in different tissues under normal conditions. During salinity stress, RPL7 was the most stable gene according to overall ranking and the best combination of reference genes was RPL7 and RNAPol II. In contrast, GAPDH was the least stable gene which was not suitable as reference genes. The study showed that different algorithms might generate inconsistent results. Therefore, the combination of several reference genes should be selected to accurately calibrate system errors. The present study was the first to select reference genes of L. maculatus by qRT-PCR and provides a useful basis for selecting the appropriate reference gene in L. maculatus. The present study also has important implications for gene expression and functional genomics research in this species or other teleost species.

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Evaluation of suitable reference genes in Brassica juncea and its wild relative Camelina sativa for qRT-PCR analysis under various stress conditions

PLoS ONE ◽

10.1371/journal.pone.0222530 ◽

2019 ◽

Vol 14 (9) ◽

pp. e0222530 ◽

Cited By ~ 2

Author(s):

Shikha Dixit ◽

Vinod Kumar Jangid ◽

Anita Grover

Keyword(s):

Brassica Juncea ◽

Reference Genes ◽

Camelina Sativa ◽

Stress Conditions ◽

Pcr Analysis ◽

Wild Relative ◽

Qrt Pcr ◽

Suitable Reference

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Identification And Evaluation of Reference Genes For Cynanchum Auriculatum Under Various Stress Conditions

10.21203/rs.3.rs-1196107/v1 ◽

2022 ◽

Author(s):

Zhi-Peng Zhu ◽

Jian-Xiang Yu ◽

Ke-Xin Wu ◽

Qin-Yi Xu ◽

Yi-Jun Kang ◽

...

Keyword(s):

Reference Genes ◽

Herbal Medicines ◽

Stress Conditions ◽

Pcr Analysis ◽

Expression Stability ◽

Qrt Pcr ◽

Chinese Herbal ◽

Cynanchum Auriculatum ◽

Screening Study ◽

Suitable Reference

Abstract Baishouwu (Cynanchum auriculatum) is a kind of critical Chinese herbal medicine. However, compared with the studies of other Chinese herbal medicines, the screening study on the reference genes of C. auriculatum is still the blank. Deterioration of the natural environment severely affects the growth and development of C. auriculatum. This study screened and identified suitable reference genes of C. auriculatum under various stress conditions. Based on qRT-PCR, geNorm, NormFinder, BestKeeper, and RefFinder were used for the expression stability evaluation of 12 potential reference genes from C. auriculatum. The ranking table showed that optimal reference genes included EF2 and SAMDC (heat stress), CYP and TUB-β (cold stress), TUB-α and GAPDH (drought stress), SAMDC and TUB-α (waterlogging stress), along with EF2 and ACT7 (salt stress). These results also demonstrated that under different abiotic stresses, suitable reference genes of plants should be selected for qRT-PCR analysis.

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Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet (Setaria italica L.) subjected to abiotic stress conditions

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-013-0335-x ◽

2013 ◽

Vol 115 (1) ◽

pp. 13-22 ◽

Cited By ~ 48

Author(s):

Karunesh Kumar ◽

Mehanathan Muthamilarasan ◽

Manoj Prasad

Keyword(s):

Abiotic Stress ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Foxtail Millet ◽

Stress Conditions ◽

Setaria Italica ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Model Plant

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Selection of suitable reference genes for qRT-PCR analysis of Begonia semperflorens under stress conditions

Molecular Biology Reports ◽

10.1007/s11033-019-05038-5 ◽

2019 ◽

Vol 46 (6) ◽

pp. 6027-6037 ◽

Cited By ~ 1

Author(s):

Yanman Li ◽

Ying Qu ◽

Yang Wang ◽

Xue Bai ◽

Geng Tian ◽

...

Keyword(s):

Reference Genes ◽

Stress Conditions ◽

Pcr Analysis ◽

Qrt Pcr ◽

Suitable Reference ◽

Selection Of ◽

Begonia Semperflorens

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Identification and validation of new reference genes for accurate quantitative reverse transcriptase-PCR normalization in the Antarctic plant Colobanthus quitensis under abiotic stress conditions

Polar Biology ◽

10.1007/s00300-021-02801-y ◽

2021 ◽

Vol 44 (2) ◽

pp. 389-405

Author(s):

Laura Bertini ◽

Silvia Proietti ◽

Francesca Focaracci ◽

Fabiana Canini ◽

Leon A. Bravo ◽

...

Keyword(s):

Abiotic Stress ◽

Reverse Transcriptase ◽

Reference Genes ◽

Vascular Plant ◽

Expression Patterns ◽

Stress Conditions ◽

Cell Physiology ◽

Colobanthus Quitensis ◽

Qrt Pcr ◽

The Antarctic

AbstractThe Antarctic ecotype of Colobanthus quitensis is a vascular plant highly adapted to the harsh environmental conditions of Maritime Antarctica which is now facing with the rapid local warming experienced in the Antarctic Peninsula during the last decades. Thus, the identification of the molecular mechanisms leading to the adaptation to this warming trend is a new target for modern cell physiology. The selection of suitable reference genes for quantification of key stress-responsive genes through quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) is important to ensure accurate and reliable results. In this study, we evaluated the expression stability of eleven candidate genes in C. quitensis under different abiotic stress conditions using geNorm and RefFinder tools. The statistical analysis showed that the appropriate reference genes varied depending on the experimental conditions, even if EF1α and PP2Acs ranked as the most stable reference genes when all stress conditions were considered. To further validate the stability of the selected reference genes, the expression patterns of C. quitensis catalase gene (CqCAT) was analyzed. The reference genes validated in this study will be useful for improving the accuracy of qRT-PCR analysis for gene expression studies of the Antarctic ecotype of C. quitensis and could be extended to other ecotypes adapted to low temperatures.

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Choice of a Stable Set of Reference Genes for qRT-PCR Analysis in Amblyomma maculatum (Acari: Ixodidae)

Journal of Medical Entomology ◽

10.1603/me12123 ◽

2012 ◽

Vol 49 (6) ◽

pp. 1339-1346 ◽

Cited By ~ 15

Author(s):

Rebecca Browning ◽

Steven Adamson ◽

Shahid Karim

Keyword(s):

Reference Genes ◽

Stable Set ◽

Pcr Analysis ◽

Amblyomma Maculatum ◽

Qrt Pcr

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Validation of reference genes for qRT-PCR analysis in peel and flesh of six apple cultivars (Malus domestica) at diverse stages of fruit development

Scientia Horticulturae ◽

10.1016/j.scienta.2018.09.033 ◽

2019 ◽

Vol 244 ◽

pp. 165-171 ◽

Cited By ~ 5

Author(s):

Lifang Zhu ◽

Chengquan Yang ◽

Yaohua You ◽

Wei Liang ◽

Nannan Wang ◽

...

Keyword(s):

Malus Domestica ◽

Fruit Development ◽

Reference Genes ◽

Pcr Analysis ◽

Qrt Pcr ◽

Apple Cultivars

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Comparison of Reliable Reference Genes Following Different Hormone Treatments by Various Algorithms for qRT-PCR Analysis of Metasequoia

International Journal of Molecular Sciences ◽

10.3390/ijms20010034 ◽

2018 ◽

Vol 20 (1) ◽

pp. 34 ◽

Cited By ~ 3

Author(s):

Jing-Jing Wang ◽

Shuo Han ◽

Weilun Yin ◽

Xinli Xia ◽

Chao Liu

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Elongation Factor ◽

Pcr Analysis ◽

Qrt Pcr ◽

Gene Normalization ◽

Accurate Normalization ◽

Suitable Reference ◽

Different Tissues ◽

Gene Expression Levels

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60β (60-kDa chaperonin β-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.

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