Validation of Reliable Reference Genes for Accurate Normalization in RT-qPCR Analysis of Codonopsis pilosula

AbstractIris germanica L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in I. germanica. In this study, eight candidate reference genes were examined for the normalization of RT-qPCR in three I. germanica cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that IgUBC and IgGAPDH were the most stable reference genes in ‘00246’ and ‘Elizabeth’, and IgTUB and IgUBC showed stable expression in ‘2010200’. IgUBC and IgGAPDH were the most stable in all samples, while IgUBQ showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of IgFT (Flowering Locus T gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in I. germanica.

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Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements

Genetics and Molecular Research ◽

10.4238/gmr16029666 ◽

2017 ◽

Vol 16 (2) ◽

Author(s):

N. Marini ◽

C.B. Bevilacqua ◽

M.V. Büttow ◽

M.C.B. Raseira ◽

S. Bonow

Keyword(s):

Reference Genes ◽

Qpcr Analysis ◽

Chilling Requirements

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

Fungal Genetics and Biology ◽

10.1016/j.fgb.2018.11.005 ◽

2019 ◽

Vol 123 ◽

pp. 33-40 ◽

Cited By ~ 2

Author(s):

Jiwei Zhang ◽

Hugh D. Mitchell ◽

Lye Meng Markillie ◽

Matthew J. Gaffrey ◽

Galya Orr ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Accurate Normalization

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Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Frontiers in Genetics ◽

10.3389/fgene.2018.00269 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 6

Author(s):

Lifeng Zhou ◽

Fengmao Chen ◽

Jianren Ye ◽

Hongyang Pan

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Developmental Stages ◽

Bursaphelenchus Mucronatus ◽

Qpcr Analysis ◽

Selection Of

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RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

FEBS Open Bio ◽

10.1002/2211-5463.12678 ◽

2019 ◽

Vol 9 (8) ◽

pp. 1403-1412 ◽

Cited By ~ 2

Author(s):

Quandong Nong ◽

Yongchao Yang ◽

Mingyong Zhang ◽

Mei Zhang ◽

Jiantong Chen ◽

...

Keyword(s):

Reference Genes ◽

Rna Seq ◽

Qpcr Analysis ◽

Selection Of

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Selection and Validation of Reference Genes for qRT-PCR in Lentinula edodes under Different Experimental Conditions

Genes ◽

10.3390/genes10090647 ◽

2019 ◽

Vol 10 (9) ◽

pp. 647 ◽

Cited By ~ 4

Author(s):

Yi Luo ◽

Gangzheng Wang ◽

Chen Wang ◽

Yuhua Gong ◽

Yinbing Bian ◽

...

Keyword(s):

Reference Gene ◽

Reference Genes ◽

Lentinula Edodes ◽

Polymerase Chain Reaction Analysis ◽

Pairwise Variation ◽

Accurate Estimation ◽

Expression Stability ◽

Experimental Conditions ◽

Qrt Pcr ◽

Accurate Normalization

Lentinula edodes is the most consumed mushroom in Asia due to its nutritional and medicinal values, and the optimal reference gene is crucial for normalization of its gene expression analysis. Here, the expression stability of 18 candidate reference genes (CRGs) in L. edodes was analyzed by three statistical algorithms (geNorm, NormFinder and BestKeeper) under different stresses (heat, cadmium excess and Trichoderma atroviride infection), different substrates (straw, sawdust and corn stalk) and different development stages (mycelia, primordia and fruit bodies). Among the 18 CRGs, 28S, Actin and α-tub exhibited the highest expression stability in L. edodes under all conditions, while GPD, SPRYP and MSF showed the least stable expression. The best reference gene in different conditions was different. The pairwise variation values showed that two genes would be sufficient for accurate normalization under different conditions of L. edodes. This study will contribute to more accurate estimation of the gene relative expression levels under different conditions using the optimal reference gene in qRT-PCR (quantitative reverse transcription polymerase chain reaction) analysis.

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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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