Towards a Systematic Validation of References in Real-Time RT-PCR

Gutierrez, Laurent; Mauriat, Mélanie; Pelloux, Jérôme; Bellini, Catherine; Wuytswinkel, Olivier Van

doi:10.1105/tpc.108.059774

Cited by 190 publications

(160 citation statements)

References 15 publications

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“…This conclusion is in agreement with recent findings that many reference genes known from vegetative tissues are not stably expressed in seeds and pollen (Paolacci et al, 2009;Chen et al, 2010;Wei et al, 2010). Gutierrez et al (2008aGutierrez et al ( , 2008b demonstrated that up to 100-fold variation could be found for expression of a target gene in distinct plant tissues and species depending on the reference gene used for normalization (i.e., there is a huge potential scope for misinterpretation of the results). They concluded that a universal reference gene does not exist but that there is an urgent need for systematic validation of reference genes based on the actual experimental conditions under investigation.…”

Section: Introductionsupporting

confidence: 80%

“…This is an often overlooked consideration, despite the vast number of publications pointing out that traditionally used reference genes are often not stably expressed under all possible circumstances, thereby highlighting the importance of validating reference genes (e.g., Volkov et al, 2003;Radonić et al, 2004;Nicot et al, 2005;Gutierrez et al, 2008b;Remans et al, 2008). Especially in the plant research field such validation is most often ignored, as noted by Gutierrez et al (2008a), who showed that out of 188 different qRT-PCR analyses recently published in leading plant biology journals only 3.2% used validated reference genes. It has been shown in a number of studies that different environmental conditions, developmental stages, tissues, or treatments strongly affect the usability of potential reference genes by causing differential expression (Thellin et al, 1999;Suzuki et al, 2000;Lee et al, 2002;Czechowski et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…We further used public microarray data sets to identify conserved expression patterns and compare the stability of reference gene expression across different Brassicaceae species. Our case study with seeds thereby provides a guideline for moving qRT-PCR work recommendations (Czechowski et al, 2005;Gutierrez et al, 2008a;Udvardi et al, 2008) beyond Arabidopsis to the cross-species level and for applying it in a spatio-temporal and hormonal manner to germinating seeds as a demanding example. The presented cross-species in silico analysis together with qRT-PCR validation in germinating seeds of L. sativum and Arabidopsis provided superior stable reference genes for use within the Brassicaceae family.…”

Section: Introductionmentioning

confidence: 99%

“…Cross-species phylotranscriptomics of distinct developmental processes such as pollen or seed development, maturation, and germination require validated cross-species reference genes for normalization of comparative qRT-PCR experiments. Here, we provide a guideline for moving qRT-PCR work recommendations (Czechowski et al, 2005;Gutierrez et al, 2008a;Udvardi et al, 2008) beyond Arabidopsis thaliana to the cross-species level illustrated in a spatio-temporal and hormonal case study with seeds for which the experimental challenges start already with the extraction of high-quality total RNA.…”

Section: Introductionmentioning

confidence: 99%

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A Guideline to Family-Wide Comparative State-of-the-Art Quantitative RT-PCR Analysis Exemplified with a Brassicaceae Cross-Species Seed Germination Case Study

et al. 2011

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Comparative biology includes the comparison of transcriptome and quantitative real-time RT-PCR (qRT-PCR) data sets in a range of species to detect evolutionarily conserved and divergent processes. Transcript abundance analysis of target genes by qRT-PCR requires a highly accurate and robust workflow. This includes reference genes with high expression stability (i.e., low intersample transcript abundance variation) for correct target gene normalization. Cross-species qRT-PCR for proper comparative transcript quantification requires reference genes suitable for different species. We addressed this issue using tissue-specific transcriptome data sets of germinating Lepidium sativum seeds to identify new candidate reference genes. We investigated their expression stability in germinating seeds of L. sativum and Arabidopsis thaliana by qRT-PCR, combined with in silico analysis of Arabidopsis and Brassica napus microarray data sets. This revealed that reference gene expression stability is higher for a given developmental process between distinct species than for distinct developmental processes within a given single species. The identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of Brassicaceae seed germination. Furthermore, using germinating seeds, we exemplify optimization of the qRT-PCR workflow for challenging tissues regarding RNA quality, transcript stability, and tissue abundance. Our work therefore can serve as a guideline for moving beyond Arabidopsis by establishing high-quality cross-species qRT-PCR.

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Section: Introductionsupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A Guideline to Family-Wide Comparative State-of-the-Art Quantitative RT-PCR Analysis Exemplified with a Brassicaceae Cross-Species Seed Germination Case Study

et al. 2011

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“…The two-step thermal cycling profile used was 15 s at 94°C and 1 min at 68°C. An eIF4A gene (At3g13920) was included in the assays as an internal control for normalizing the variations in cDNA amounts used (39). qRT-PCR was carried out in biological triplicates using total RNA samples extracted from three independent plant materials grown or treated under identical conditions.…”

Section: Methodsmentioning

confidence: 99%

The E3 Ubiquitin Ligase HOS1 Regulates Arabidopsis Flowering by Mediating CONSTANS Degradation Under Cold Stress

Jung

Seo

Park

2012

Journal of Biological Chemistry

104

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Background: Intermittent cold stress delays flowering. This results from interaction between the cold and photoperiodic pathways. Results: CO protein is degraded through an HOS1-mediated ubiquitination mechanism during brief cold treatments. Conclusion: CO acts as a molecular hub that integrates photoperiod and cold signals into the flowering genetic pathways. Significance: The CO-HOS1 module is crucial for fine-tuning of photoperiodic flowering under short term temperature fluctuations.

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The optimal reference gene validation in Cyclocarya paliurus (Batal.) Iljinskaja under environmental stresses

et al. 2022

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Cyclocarya paliurus (Batal.) Iljinskaja is a special plant species grown at high elevation in southwestern China. The bark, leaves, and roots are used for many purposes including reduction of fever and enhancement of blood insulin level. However, its growth is usually limited by the fragile environment and climate variability. Previous studies attributed the environment‐relevant yield reductions to stress induced gene expression changes. The accuracy and reliability of reverse transcription quantitative polymerase chain reaction (RT‐qPCR), a commonly accepted gene expression method, depends on the choice of appropriate reference genes, which are used for gene expression normalization. However, there are no reports at present about reference genes of C. paliurus under environmental stresses. Therefore, this study was conducted to identify reference genes for C. paliurus under environmental stresses. In this work, we selected seven common reference genes as our candidates: ACT2, ACTF, GAPDH, TUA, TUB, UBQ4, and 18SrRNA. The stability of these seven reference genes in C. paliurus was quantified by RT‐qPCR and evaluated by geNorm and NormFinder algorithms. The results suggest that although there were variations of the stability of the internal reference genes, two of them were usually good enough for the normalization purpose for low temperature (4 °C), high salinity (NaCl), and drought stress (polyethylene glycol [PEG] treatment). However, these genes should not be used for high‐temperature (42 °C) stress. This study provides insight for reference gene selection under experimental stresses in C. paliurus. It also sets the foundation of wide and accurate use of RT‐qPCR under environmental stresses.

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Towards a Systematic Validation of References in Real-Time RT-PCR

Cited by 190 publications

References 15 publications

A Guideline to Family-Wide Comparative State-of-the-Art Quantitative RT-PCR Analysis Exemplified with a Brassicaceae Cross-Species Seed Germination Case Study

A Guideline to Family-Wide Comparative State-of-the-Art Quantitative RT-PCR Analysis Exemplified with a Brassicaceae Cross-Species Seed Germination Case Study

The E3 Ubiquitin Ligase HOS1 Regulates Arabidopsis Flowering by Mediating CONSTANS Degradation Under Cold Stress

The optimal reference gene validation in Cyclocarya paliurus (Batal.) Iljinskaja under environmental stresses

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