The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription‐polymerase chain reaction (RT‐PCR) analysis in plants

Gutierrez, Laurent; Mauriat, Mélanie; Guénin, Stéphanie; Pelloux, Jérôme; Lefebvre, Jean‐François; Louvet, Romain; Rustérucci, Christine; Moritz, Thomas; Guérineau, François; Bellini, Catherine; Wuytswinkel, Olivier Van

doi:10.1111/j.1467-7652.2008.00346.x

Cited by 634 publications

(563 citation statements)

References 25 publications

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“…The twostep thermal cycling profile used was 15 s at 94 °C and 1 min at 68 °C. An eIF4A gene (At3g13920) was included in the reactions as an internal control for normalizing the variations in cDNA amounts used 25 . All qRT-PCR reactions were carried out in biological triplicates using RNA samples extracted from three independent plant materials grown under identical growth conditions.…”

Section: Methodsmentioning

confidence: 99%

Two splice variants of the IDD14 transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism

et al. 2011

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Section: Methodsmentioning

confidence: 99%

Two splice variants of the IDD14 transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism

et al. 2011

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“…This made the evaluation of several reference genes for qRT-PCR imperative (Gutierrez et al, 2008). After evaluating five possible genes as suggested by Reid et al (2006;Supplemental Table S2), we determined that the average relative transcriptional abundance of genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and SAND family protein (SAND) was the most constant and did not differ statistically across the berry developmental stages (Supplemental Fig.…”

Section: Pa Biosynthetic Genes Are Expressed Very Early In Blueberry mentioning

confidence: 99%

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism

Zifkin

Jin²,

Ozga³

et al. 2011

Plant Physiology

256

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Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative realtime reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3#-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3#5#-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis.Highbush blueberry (Vaccinium corymbosum; Ericaceae) is one of the most economically important fruit crops in North America. Blueberry fruit have been the focus of much recent attention due to numerous reports of their positive effects on human health. These benefits are generally attributed to high levels of polyphenolics, in particular the flavonoids (Rasmussen et al., 2005). Highbush blueberries have one of the highest in vitro antioxidant capacities of any fruit or vegetable (Prior and Gu, 2005;Wu et al., 2006). The major health benefits linked to the consumption of blueberries include a reduced risk for cardiovascular (Basu et al., 2010) and neurodegenerative (Neto, 2007) diseases. Furthermore, experiments on rodents suggest that blueberry extracts may also prevent cancer, slow tumor growth, and reverse cognitive and behavioral deficits related to stroke and aging (Lau et al., 2005;Gordillo et al., 2009).The three common types of flavonoids that accumulate in blueberry fruit are the flavonols, anthocya-

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“…C_LG_XV0407; GenBank accession no. CA825222) and a putative protein (Gutierrez et al, 2008; P. trichocarpa gene model estExt_fgenesh4_pm. C_LG_IX0344) as reference genes.…”

Section: Nimblegen Microarray Transcript Profilingmentioning

confidence: 99%

The Ectomycorrhizal FungusLaccaria bicolorStimulates Lateral Root Formation in Poplar and Arabidopsis through Auxin Transport and Signaling

et al. 2009

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The early phase of the interaction between tree roots and ectomycorrhizal fungi, prior to symbiosis establishment, is accompanied by a stimulation of lateral root (LR) development. We aimed to identify gene networks that regulate LR development during the early signal exchanges between poplar (Populus tremula 3 Populus alba) and the ectomycorrhizal fungus Laccaria bicolor with a focus on auxin transport and signaling pathways. Our data demonstrated that increased LR development in poplar and Arabidopsis (Arabidopsis thaliana) interacting with L. bicolor is not dependent on the ability of the plant to form ectomycorrhizae. LR stimulation paralleled an increase in auxin accumulation at root apices. Blocking plant polar auxin transport with 1-naphthylphthalamic acid inhibited LR development and auxin accumulation. An oligoarray-based transcript profile of poplar roots exposed to molecules released by L. bicolor revealed the differential expression of 2,945 genes, including several components of polar auxin transport (PtaPIN and PtaAUX genes), auxin conjugation (PtaGH3 genes), and auxin signaling (PtaIAA genes). Transcripts of PtaPIN9, the homolog of Arabidopsis AtPIN2, and several PtaIAAs accumulated specifically during the early interaction phase. Expression of these rapidly induced genes was repressed by 1-naphthylphthalamic acid. Accordingly, LR stimulation upon contact with L. bicolor in Arabidopsis transgenic plants defective in homologs of these genes was decreased or absent. Furthermore, in Arabidopsis pin2, the root apical auxin increase during contact with the fungus was modified. We propose a model in which fungus-induced auxin accumulation at the root apex stimulates LR formation through a mechanism involving PtaPIN9-dependent auxin redistribution together with PtaIAA-based auxin signaling.Most temperate forest trees develop a mutualistic root symbiosis with ectomycorrhizal soil fungi. During the establishment of ectomycorrhiza (ECM), fungal hyphae invade the root from root cap cells in and upwards to the epidermis (Horan et al., 1988). After attachment to epidermal cells, hyphae multiply to form a series of layers that differentiate to establish a mantle structure around the root (Horan et al., 1988). An internal network of hyphae between the epidermis and root cortex cells forms the Hartig net (Blasius et al., 1986), while extraradical hyphae prospect throughout the surrounding soil and gather nutrients. Morphological observations of forming ECMs have shown that in the vicinity of the fungus the root architecture of the host plant is profoundly modified. Interaction with hyphae stimulates lateral root (LR) formation, dichotomy of the root apical meristem in conifer species, and cytodifferentiation of root cells (radial elongation and root hair decay; Horan et al., 1988;Dexheimer and Pargney, 1991;. Even though several studies have focused on LR stimulation during ECM formation, it still remains unclear what the molecular mechanisms are that modify root development during contact with the fungus.In the ...

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The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription‐polymerase chain reaction (RT‐PCR) analysis in plants

Abstract: SummaryReverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably

Cited by 634 publications

References 25 publications

Two splice variants of the IDD14 transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism

Two splice variants of the IDD14 transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism

The Ectomycorrhizal FungusLaccaria bicolorStimulates Lateral Root Formation in Poplar and Arabidopsis through Auxin Transport and Signaling

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