Reference genes for accurate normalization of gene expression in wood-decomposing fungi

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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Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses

Gene ◽

10.1016/j.gene.2014.10.049 ◽

2015 ◽

Vol 554 (2) ◽

pp. 205-214 ◽

Cited By ~ 61

Author(s):

Vanessa Galli ◽

Joyce Moura Borowski ◽

Ellen Cristina Perin ◽

Rafael da Silva Messias ◽

Julia Labonde ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Quantitative Pcr ◽

Reference Genes ◽

Real Time Quantitative Pcr ◽

Accurate Normalization

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Identification and Selection of Reference Genes for Quantitative Transcript Analysis in Corydalis yanhusuo

Genes ◽

10.3390/genes11020130 ◽

2020 ◽

Vol 11 (2) ◽

pp. 130 ◽

Cited By ~ 1

Author(s):

Zhenzhen Bao ◽

Kaidi Zhang ◽

Hanfeng Lin ◽

Changjian Li ◽

Xiurong Zhao ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Elongation Factor ◽

High Sensitivity ◽

Future Research ◽

Polypyrimidine Tract Binding Protein ◽

Accurate Normalization ◽

Ubiquitin Conjugating Enzyme ◽

Protein Phosphatase 2 ◽

Selection Of

Corydalis yanhusuo is a medicinal plant frequently used in traditional Chinese medicine, which has effective medical effects in many aspects. Real-time polymerase chain reaction (RT-PCR) has been one of the most widely used methods in biosynthesis research due to its high sensitivity and quantitative properties in gene expression analysis. To obtain accurate normalization, reference genes are often selected in advance; however, no reference genes are available in C. yanhusuo. Herein, 12 reference gene candidates, named cyclophilin 2 (CYP2), elongation factor 1-α (EF1-α), protein phosphatase 2 (PP2A), SAND protein family (SAND), polypyrimidine tract-binding protein (PTBP), TIP41-like protein (TIP41), lyceraldehyde-3-phosphate hydrogenase (GAPDH), ubiquitin-conjugating enzyme 9 (UBC9), cyclophilin 1 (CYP1), tubulin beta (TUBA), thioredoxin (YLS8), and polyubiquitin 10 (UBQ10), were selected for stability analysis. After being treated with hormone, UV, salt, metal, oxidative, drought, cold (4 °C), and hot stresses (40 °C), the qRT-PCR data of the selected genes was analyzed with NormFinder, geNorm, and BestKeeper. The result indicated that GAPDH, SNAD, and PP2A were the top three most stable reference genes under most treatments. This study selected and validated reliable reference genes in C. yanhusuo under various environmental conditions, which can provide great help for future research on gene expression normalization in C. yanhusuo.

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Validation of reference genes for accurate normalization by quantitative polymerase chain reaction in sugarcane drought stress studies using two cultivars

Ciência Rural ◽

10.1590/0103-8478cr20180152 ◽

2018 ◽

Vol 48 (11) ◽

Author(s):

Dennis Crystian ◽

Jackeline Terto ◽

José Vieira Silva ◽

Cícero Almeida

Keyword(s):

Gene Expression ◽

Polymerase Chain Reaction ◽

Drought Stress ◽

Reference Genes ◽

Quantitative Polymerase Chain Reaction ◽

Elongation Factor ◽

Chain Reaction ◽

Stress Studies ◽

Accurate Normalization ◽

Polymerase Chain

ABSTRACT: The aim of this study was to analyze the expression of putative reference genes in sugarcane under drought stress. The varieties RB72454 and RB72910 were cultivated and the treatments control and drought stress applied to 135-day-old plants grown under field conditions. The stress level of the plants was measured by rate of photosynthesis, transpiration, and stomatal conductance. For each biological replicate, expression analyses were conducted using quantitative polymerase chain reaction for the genes α-tubulin, β-tubulin, β-actin, cyclophilin, eukaryotic elongation factor 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H3 and ubiquitin. Stability was evaluated using the geNorm, NormFinder, and BestKeeper software packages. Among the candidate genes, GAPDH was identified as the most stable in all software, indicating its suitability for gene expression studies in sugarcane undergoing drought stress; the gene β-actin was the second most stable. These findings suggest using GAPDH and β-actin for normalization in relative gene expression in sugarcane.

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