Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR

Kianianmomeni, Arash; Hallmann, Armin

doi:10.1007/s11033-013-2784-z

Cited by 27 publications

(23 citation statements)

References 47 publications

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“…Nevertheless, in our study, ACT failed to be ranked as a satisfactory reference gene under most of the experimental conditions except biotic stress, and even yielded the poorest values during drought and salinity stress. On the other hand, 18S rRNA was previously proved to be one of the best reference genes for L. brownii and some other plant species (Kianianmomeni & Hallmann, 2013; Luo et al, 2014; Saha & Vandemark, 2013), but in our study, 18S rRNA was generally evaluated as the one of the least stable reference genes except under drought stress. This is possibly caused by their genotype differences.…”

Section: Discussionmentioning

confidence: 61%

Evaluation of putative reference genes for quantitative real-time PCR normalization inLilium regaleduring development and under stress

Liu

Chi

Zhang

et al. 2016

PeerJ

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Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression, Lilium, and particularly L. regale, has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely 18S rRNA, ACT, BHLH, CLA, CYP, EF1, GAPDH, SAND and TIP41, were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic (Botrytis elliptica), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms, BHLH was superior to the other candidates when all the experimental treatments were analyzed together; CLA and EF1 were also recommended by two of the three algorithms. As for specific conditions, EF1 under various developmental stages, SAND under biotic stress, CYP/GAPDH under drought stress, and TIP41 under salinity stress were generally considered suitable. All the algorithms agreed on the stability of SAND and GAPDH under cold stress, while only CYP was selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level of LrLOX in leaves inoculated with B. elliptica. Our study would be beneficial for future studies on gene expression and molecular breeding of Lilium.

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Section: Discussionmentioning

confidence: 61%

Evaluation of putative reference genes for quantitative real-time PCR normalization inLilium regaleduring development and under stress

Liu

Chi

Zhang

et al. 2016

PeerJ

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“…Reference gene identification and selection has been conducted in a variety of plant species, however, studies are limited in non-vascular plants to the moss Physcomitrella patens ( Le Bail et al, 2013 ) and the brown algae Ectocarpus siliculosus ( Kianianmomeni and Hallmann, 2013 ). To date, no study has been performed to evaluation stable reference gene for a desiccation tolerant moss species in response to abiotic stress.…”

Section: Introductionmentioning

confidence: 99%

Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration

Zhang

et al. 2015

Front. Plant Sci.

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Syntrichia caninervis is the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert. The extreme desert environment is characterized by prolonged drought, temperature extremes, high radiation and frequent cycles of hydration and dehydration. S. caninervis is an ideal organism for the identification and characterization of genes related to abiotic stress tolerance. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) expression analysis is a powerful analytical technique that requires the use of stable reference genes. Using available S. caninervis transcriptome data, we selected 15 candidate reference genes and analyzed their relative expression stabilities in S. caninervis gametophores exposed to a range of abiotic stresses or a hydration-desiccation-rehydration cycle. The programs geNorm, NormFinder, and RefFinder were used to assess and rank the expression stability of the 15 candidate genes. The stability ranking results of reference genes under each specific experimental condition showed high consistency using different algorithms. For abiotic stress treatments, the combination of two genes (α-TUB2 and CDPK) were sufficient for accurate normalization. For the hydration-desiccation-rehydration process, the combination of two genes (α-TUB1 and CDPK) were sufficient for accurate normalization. 18S was among the least stable genes in all of the experimental sets and was unsuitable as reference gene in S. caninervis. This is the first systematic investigation and comparison of reference gene selection for RT-qPCR work in S. caninervis. This research will facilitate gene expression studies in S. caninervis, related moss species from the Syntrichia complex and other mosses.

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“…The products of all real-time RT-PCR reactions were visualized using agarose gel electrophoresis to assure amplification of a single product of the correct size. The specific primers were as follows: 5′-CCTGAGCCACATCAAGTGCAC and 5′-GGATGTCCACGATGGCCTCG for amplification of a fragment of the luciferase ( g-luc ) transcript from G. princeps (codon-optimized for C. reinhardtii ) [ 33 ] (expected cDNA length: 119 bp), and 5′-GTAGTGGCTACTGTGAATCTGG and 5′-GCTCTCTAATACGCATAATGACG for amplification of a fragment of the TATA-box binding protein transcript tbp A [ 61 ] (expected cDNA length: 118 bp), which has been established as a reference gene for quantitative gene expression studies in V. carteri [ 62 ]. The cycling conditions in the CFX96 Touch™ Real-Time PCR Detection System were as follows: reverse transcription was at 50°C for 30 min followed by polymerase activation at 95°C for 2 min and 40 cycles of DNA amplification with 95°C for 5 s, 55°C for 10 s and 72°C for 5 s. Melting curves were recorded to check for amplification of a single specific product.…”

Section: Methodsmentioning

confidence: 99%

The inducible nitA promoter provides a powerful molecular switch for transgene expression in Volvox carteri

et al. 2015

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BackgroundThe multicellular green alga Volvox carteri represents an attractive model system to study various aspects of multicellularity like cellular differentiation, morphogenesis, epithelial folding and ECM biogenesis. However, functional and molecular analyses of such processes require a wide array of molecular tools for genetic engineering. So far there are only a limited number of molecular tools available in Volvox.ResultsHere, we show that the promoter of the V. carteri nitrate reductase gene (nitA) is a powerful molecular switch for induction of transgene expression. Strong expression is triggered by simply changing the nitrogen source from ammonium to nitrate. We also show that the luciferase (g-luc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of the unicellular alga Chlamydomonas reinhardtii, is a suitable reporter gene in V. carteri. Emitted light of the chemiluminescent reaction can be easily detected and quantified with a luminometer. Long-term stability of inducible expression of the chimeric nitA/g-luc transgenes after stable nuclear transformation was demonstrated by transcription analysis and bioluminescence assays.ConclusionTwo novel molecular tools for genetic engineering of Volvox are now available: the nitrate-inducible nitA promoter of V. carteri and the codon-adapted luciferase reporter gene of G. princeps. These novel tools will be useful for future molecular research in V. carteri.

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Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR

Cited by 27 publications

References 47 publications

Evaluation of putative reference genes for quantitative real-time PCR normalization inLilium regaleduring development and under stress

Evaluation of putative reference genes for quantitative real-time PCR normalization inLilium regaleduring development and under stress

Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration

The inducible nitA promoter provides a powerful molecular switch for transgene expression in Volvox carteri

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