Evaluation of reference genes for quantitative real-time PCR in Ostertagia ostertagi by the coefficient of variation and geNorm approach

Zeveren, A.M. Van; Visser, Aline; Hoorens, Prisca; Vercruysse, Jozef; Claerebout, Edwin; Geldhof, Peter

doi:10.1016/j.molbiopara.2007.03.005

Cited by 18 publications

(15 citation statements)

References 8 publications

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“…In the case of parasitic nematodes, transcription stability of housekeeping genes has been investigated in Brugia malayi (Li et al, 2004), Ancylostoma caninum (Trivedi and Arasu, 2005), and, more recently, in Ostertagia ostertagi (Van Zeveren et al, 2007). Li et al (2004) analyzed transcription stability of different housekeeping genes in adult male and female worms and assigned genes with approximately identical Ct (threshold cycle) values as equally transcribed.…”

Section: Introductionmentioning

confidence: 99%

“…However, both the Ct comparison and CV analysis disregard changes in the transcriptome due to quantitative variations in the repertoire of transcripts produced by different developmental stages of the parasite. For this reason, Van Zeveren et al (2007) evaluated O. ostertagi housekeeping genes using a program named geNorm following the CV analysis of Ct values. The geNorm software relies on the principle that the transcription ratio of two ideal reference genes is identical in all samples, regardless of experimental or developmental influences.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus

Strübe

Buschbaum

Wolken

et al. 2008

Gene

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus

Strübe

Buschbaum

Wolken

et al. 2008

Gene

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“…Target genes are ABC transporter subfamily A5 (ABCA5), glutathione-s-transferase (GST), and ATP synthase lipid-binding protein (ATP5G3). For normalization factors, refer to "Materials and methods" Zeveren et al 2007). To confirm that the selection of reference genes is important, we compared four normalization factors: quantities of the three single reference genes ACTB, PGK, and snRNP or the geometric mean of quantities of the three reference genes ACTB, PGK, and CAPS.…”

Section: Discussionmentioning

confidence: 99%

Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR

Yoo

Kim

et al. 2008

Parasitol Res

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The accuracies of relative gene expressions as determined by quantitative real-time polymerase chain reaction are largely dependent on the variabilities of the reference genes used. Validation of the stabilities of reference genes under experimental conditions is an essential initial step for comparative studies on the expression levels of target genes in experimental groups. Using three total RNA samples extracted independently from Clonorchis sinensis metacercariae and adults, we determined the gene expression stabilities of eight reference gene candidates and the relative transcript levels of three target genes using the geNorm program. The reference genes found to be stably expressed in metacercariae and adults were phosphoglycerate kinase, beta-actin, and calcyphosine; reference genes found to be stably expressed under gamma-irradiated and non-irradiated conditions were succinate dehydrogenase, small nuclear ribonucleoprotein, and beta-actin; and those stably expressed regardless of bile treatment were small nuclear ribonucleoprotein, phosphoglycerate kinase, and succinate dehydrogenase. According to our data, the expression levels of target genes are dependent on normalization factors, such as the C (T) values of single reference genes and the geometric mean of the C (T) values of three reference genes. When comparing C. sinensis gene expressions, we propose to employ the geometric mean of the C (T) values of more than three reference genes validated in the same experimental setting.

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“…The RNA extraction, the cDNA synthesis, the choice of housekeeping genes and the normalisation of the raw data were performed as described in Van Zeveren et al (2007). The reaction mixture (20 ll) consisted of a master-mix containing Taq DNA polymerase, deoxynucleotide triphosphatases (dNTPs) mixture, SYBR green I, 2 or 3 mM MgCl 2 , 0.5 lM of each primer (sequences available on request) and 2 ll of template (cDNA or plasmid DNA).…”

Section: Quantitative and Semi-quantitative Pcrmentioning

confidence: 99%

“…The RNA extraction and cDNA synthesis was described by Van Zeveren et al (2007). A standard PCR reaction (Invitrogen) (12.5 ll total volume) with 0.5 ll of cDNA (Van Zeveren et al, 2007) of the different life-cycle stages of O. ostertagi was performed with 32 cycles of amplification. To correct for sample-to-sample variation during RNA isolation and reverse transcription step, the expression profile of the reference gene rpl13 was also examined by PCR.…”

Section: Quantitative and Semi-quantitative Pcrmentioning

confidence: 99%

Gender-enriched transcription of activation associated secreted proteins in Ostertagia ostertagi

Visser

Zeveren

Meyvis

et al. 2008

International Journal for Parasitology

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Activation associated secreted proteins (ASP) are members of a nematode-specific protein family belonging to the SCP/Tpx-1/Ag5/ PR-1/Sc7 family. Three different types of molecules have been identified in this family: two-domain ASPs and single-domain ASPs showing homology to either the C-terminal or N-terminal domain of the two-domain ASP. The function of these proteins is still unclear, but a role in transition to parasitism and a role as allergen are often suggested. Here we report that the abomasal cattle parasite Ostertagia ostertagi produces at least 15 ASPs, including two-domain and C-and N-type single-domain ASPs. Ten of these are highly transcribed in the L4 stage, whereas others are highly enriched in adult male worms. The latter was especially the case for the N-type single-domain ASPs Oo-ASP1 and Oo-ASP2 and also for Oo-ASP3, which is homologous with the Haemonchus contortus and Ancylostoma caninum Ctype single-domain ASPs. Immunohistochemistry showed that Oo-ASP3 was localised in the oesophagus. Oo-ASP1 and Oo-ASP2 on the other hand were localised in the reproductive tract of both male and female worms, suggesting a role in reproduction or in the development of the reproductive tract. Ó

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Evaluation of reference genes for quantitative real-time PCR in Ostertagia ostertagi by the coefficient of variation and geNorm approach

Cited by 18 publications

References 8 publications

Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus

Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus

Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR

Gender-enriched transcription of activation associated secreted proteins in Ostertagia ostertagi

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