Housekeeping Gene Selection for Real Time-PCR Normalization in Female Hop (Humulus lupulus L) Tissues

Maloukh, Lina; Matoušek, J.; Bockstaele, Erik Van; Roldán‐Ruiz, Isabel

doi:10.1007/bf03263295

Cited by 21 publications

(15 citation statements)

References 25 publications

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“…The expression stability differences might be a result of different tissues analyzed in the compared experiments. The second observed difference relates to the ranking of the stability of the reference genes, e.g., GAPDH was found to be one of the two most stable genes in a previous study [21] but to be one of the least stable genes in our study. The regulation of reference genes is not only variety/cultivar specific but may also be tissue specific and influenced by the experimental conditions [29]; [30]; [11].…”

Section: Discussioncontrasting

confidence: 64%

“…Comparing our study to the study of Maloukh et al [21], the stability values of four out of six reference genes ( NADH , 7SLRNA , DRH1 , GAPDH ) were significantly lower in our experiment; for example, DRH1 had a 7.5 times lower stability value in our experiment, although both experiments indicated that it is one of the most stable reference genes. The expression stability differences might be a result of different tissues analyzed in the compared experiments.…”

Section: Discussioncontrasting

confidence: 51%

“…The majority of gene expression studies on hop have been performed using only one housekeeping gene [18]; [19]; [20]; only in the most recent study were 6 housekeeping genes tested across different hop tissues of female genotypes [21]. Comparing our study to the study of Maloukh et al [21], the stability values of four out of six reference genes ( NADH , 7SLRNA , DRH1 , GAPDH ) were significantly lower in our experiment; for example, DRH1 had a 7.5 times lower stability value in our experiment, although both experiments indicated that it is one of the most stable reference genes.…”

Section: Discussionmentioning

confidence: 99%

“…Primer sequences and the names of the genes are given in Table 1. We also used two published primers of the genes 7SLRNA (7SL component of the signal recognition particle) and DRH1 (DEAD box RNA helicase), which have been recently used as internal references in hop [21] (Table 1). …”

Section: Methodsmentioning

confidence: 99%

“…In studies of gene expression in hop, the normalization strategy has mainly used only one housekeeping gene: the transcript levels of the O-methyltransferase gene were normalized by using GAPDH as the housekeeping gene [18], the expression levels of Valerophenone Synthase were normalized by the gene encoding the polyubiquitin protein [19] and the expression of HlMyb3 transcription factor was normalized by the 7SL-RNA reference gene [20]. In the most recent study of hop, 6 housekeeping genes were tested across different hop tissues of female genotypes and three of them ( GAPDH, DRH1, 7SI-RNA ) were used for normalization of expression of Hua enhancer 1 transcript factor [21].…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of Reference Genes for RT-qPCR Expression Studies in Hop (Humulus lupulus L.) during Infection with Vascular Pathogen Verticillium albo-atrum

2013

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Hop plant (Humulus lupulus L.), cultivated primarily for its use in the brewing industry, is faced with a variety of diseases, including severe vascular diseases, such as Verticillium wilt, against which no effective protection is available. The understanding of disease resistance with tools such as differentially expressed gene studies is an important objective of plant defense mechanisms. In this study, we evaluated twenty-three reference genes for RT-qPCR expression studies on hop under biotic stress conditions. The candidate genes were validated on susceptible and resistant hop cultivars sampled at three different time points after infection with Verticillium albo-atrum. The stability of expression and the number of genes required for accurate normalization were assessed by three different Excel-based approaches (geNorm v.3.5 software, NormFinder, and RefFinder). High consistency was found among them, identifying the same six best reference genes (YLS8, DRH1, TIP41, CAC, POAC and SAND) and five least stably expressed genes (CYCL, UBQ11, POACT, GAPDH and NADH). The candidate genes in different experimental subsets/conditions resulted in different rankings. A combination of the two best reference genes, YLS8 and DRH1, was used for normalization of RT-qPCR data of the gene of interest (PR-1) implicated in biotic stress of hop. We outlined the differences between normalized and non-normalized values and the importance of RT-qPCR data normalization. The high correlation obtained among data standardized with different sets of reference genes confirms the suitability of the reference genes selected for normalization. Lower correlations between normalized and non-normalized data may reflect different quantity and/or quality of RNA samples used in RT-qPCR analyses.

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