Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

Li, Qingdi Quentin; Skinner, Jeff; Bennett, John E.

doi:10.1186/1471-2199-13-22

Cited by 74 publications

(64 citation statements)

References 54 publications

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“…For example, ACT1 exhibited a significantly lower expression level than the other candidates in Saccharomyces cerevisiae under sulfite stress (Nadai, Campanaro, Giacomini, & Corich, ), and the variation of act expressions in Trichoderma reesei was detected when incubated under different conditions (Steiger, Mach, & Mach‐Aigner, ). GAPDH was also found to be unstable during azole treatment in Candida glabrata (Li, Skinner, & Bennett, ). Therefore, new reference genes that exhibit stable and high expression levels under specific conditions should be considered.…”

Section: Discussionmentioning

confidence: 99%

“…HIS, Histone; ACT, Actin; EF1, Elongation factor 1; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; RPP, RNA polymerase II CTD phosphatase; UBQ, Ubiquitin; UCE, Ubiquitinconjugating enzyme; TBP, TATA-binding protein; SSD, Succinatesemialdehyde dehydrogenase F I G U R E 1 Verification of primer specificity of nine candidate reference genes from Clonostachys rosea 67-1. M: Marker; 1: Actin; 2: Elongation factor 1; 3: Glyceraldehyde-3-phosphate dehydrogenase; 4: Histone; 5: RNA polymerase II CTD phosphatase; 6: Succinatesemialdehyde dehydrogenase; 7: TATA-binding protein; 8: Ubiquitin; 9: Ubiquitin-conjugating enzyme glabrata (Li, Skinner, & Bennett, 2012). Therefore, new reference genes that exhibit stable and high expression levels under specific conditions should be considered.…”

Section: Discussionmentioning

confidence: 99%

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Identification of suitable reference genes during the formation of chlamydospores in Clonostachys rosea 67‐1

Zhang

Sun

et al. 2017

MicrobiologyOpen

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Clonostachys rosea is a potential biocontrol fungus that can produce highly resistant chlamydospores under specific conditions. To investigate the genes related to chlamydospore formation, we identified reliable reference genes for quantification of gene expression in C. rosea 67‐1 during sporulation. In this study, nine reference genes, actin (ACT), elongation factor 1 (EF1), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), histone (HIS), RNA polymerase II CTD phosphatase Fcp1 (RPP), succinate‐semialdehyde dehydrogenase (SSD), TATA‐binding protein (TBP), ubiquitin (UBQ), and ubiquitin‐conjugating enzyme (UCE), were selected and cloned from 67‐1, and their expression stability during chlamydospore formation was determined using reverse transcription quantitative PCR and assessed using the software geNorm, NormFinder and BestKeeper. The Ct values of the candidates ranged from 19.9 to 29.7, among which HIS,ACT and SSD exhibited high expression levels. The statistical analysis showed that ACT and SSD were most stably expressed, while UBQ and GAPDH showed relatively large variations under different culture conditions. Calculation of pairwise variation value indicated that two reference genes were required for precise quantification. Finally, ACT and SSD were selected to normalize gene expression during chlamydospore production in C. rosea 67‐1. To the best of our knowledge, this is the first report of SSD as a reference gene. This study will facilitate the accurate quantification of differentially expressed genes during the generation of chlamydospores and contribute to the investigation of the molecular mechanism underlying chlamydospore formation in C. rosea.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Identification of suitable reference genes during the formation of chlamydospores in Clonostachys rosea 67‐1

Zhang

Sun

et al. 2017

MicrobiologyOpen

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“…Conditions of q-RT-PCR were as reported [13]. Data were analyzed by using the software provided by ABI Company (Applied Biosystems, Foster City, USA) and calculated by the 2 –ΔΔCt method, according to previous studies [48,49]. …”

Section: Methodsmentioning

confidence: 99%

Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids

Wang

Lin

et al. 2013

BMC Plant Biol

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BackgroundGenetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood.ResultsWe investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal F1 hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. While significant genotypic difference was observed in frequencies of both types of alterations, only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1 hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations.ConclusionsOur results, based on molecular marker analysis of ca. 1,000 genomic loci, document that genetic alteration is the major cause of somaclonal variation in rice, which is concomitant with epigenetic alterations. Perturbed expression by tissue culture of a set of 41 genes encoding for enzymes involved in DNA repair and DNA methylation is associated with both genetic and epigenetic alterations. There exist fundamental differences among distinct genotypes, pure-lines, hybrids and tetraploids, in propensities of generating both genetic and epigenetic alterations under the tissue culture condition. Parent-of-origin has a conspicuous effect on the alteration frequencies.

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“…Threshold cycles of the target were normalized to the C T of the CgPGK1gene (DC T = C T Target ÀC T CgPGK1 ) . (Li et al, 2012). Therefore, the negative DDC T values are equivalent to log 2 relative fold changes.…”

Section: Rt-qpcrmentioning

confidence: 99%

Milbemycin A4 oxime as a probe of azole transport inCandida glabrata

et al. 2014

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Azole resistance in Candida glabrata, a pathogenic yeast, has prompted studies of compounds that have therapeutic potential by reversing azole resistance. Milbemycin A4 oxime blocked azole efflux and enhanced azole susceptibility four-fold in 28 clinical isolates of C. glabrata. Specificity of the milbemycin A4 oxime effect depended on the drug transporter and the substrate being effluxed. The major effect of milbemycin A4 oxime was inhibition of azole and rhodamine 6G efflux by the ATP-Binding Cassette (ABC) transporters CgCDR1 and PDH1. Milbemycin A4 oxime effect did not extend to oligomycin, transported by the ABC transporter YOR1 or to benomyl, transported by the Major Facilitator Superfamily transporter, CgFLR1. Milbemycin A4 oxime did not suppress transcription of CgCDR1 but increased CgCDR1 expression 126-fold. Selectivity of the effect is compatible with the concept that milbemycin A4 oxime may interact directly with a one or more drug-binding sites of the major azole transporters.

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Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

Cited by 74 publications

References 54 publications

Identification of suitable reference genes during the formation of chlamydospores in Clonostachys rosea 67‐1

Identification of suitable reference genes during the formation of chlamydospores in Clonostachys rosea 67‐1

Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids

Milbemycin A4 oxime as a probe of azole transport inCandida glabrata

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