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Leukocyte count affects expression of reference genes in canine whole blood samples.

Piek CJ, Brinkhof B, Rothuizen J, Dekker A, Penning LC - BMC Res Notes (2011)

Bottom Line: The disease category and the white blood cell count significantly affected reference gene expression.The discrepancy between the ranking of reference genes in this study by Normfinder and Genorm can be explained by differences between the experimental groups such as "disease category" and "WBC count".This stresses the importance of assessing the expression stability of potential reference genes for gene experiments in canine whole blood anew for each specific experimental condition.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Sciences of Companion Animals, Utrecht, Utrecht University, PO Box 80154, 3508 TD Utrecht, The Netherlands. C.J.Piek@uu.nl.

ABSTRACT

Background: The dog is frequently used as a model for hematologic human diseases. In this study the suitability of nine potential reference genes for quantitative RT-PCR studies in canine whole blood was investigated.

Findings: The expression of these genes was measured in whole blood samples of 263 individual dogs, representing 73 different breeds and a group of 40 mixed breed dogs, categorized into healthy dogs and dogs with internal and hematological diseases, and dogs that underwent a surgical procedure. GeNorm analysis revealed that a combination of 5 to 6 of the most stably expressed genes constituted a stable normalizing factor. Evaluation of the expression revealed different ranking of reference genes in Normfinder and GeNorm. The disease category and the white blood cell count significantly affected reference gene expression.

Conclusions: The discrepancy between the ranking of reference genes in this study by Normfinder and Genorm can be explained by differences between the experimental groups such as "disease category" and "WBC count". This stresses the importance of assessing the expression stability of potential reference genes for gene experiments in canine whole blood anew for each specific experimental condition.

No MeSH data available.


Related in: MedlinePlus

Pairwise variations between 2 sequential normalization factors including an increasing number of potential reference genes . To determine the optimal number of reference genes, first the geometric mean of the expression of the previously ranked genes was calculated and then pair wise variations between sequential normalisation factors were calculated. Using the cut-off recommended by GeNorm of 0.15 the optimal number of reference genes for the data set in this study would be at least 5.
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Figure 2: Pairwise variations between 2 sequential normalization factors including an increasing number of potential reference genes . To determine the optimal number of reference genes, first the geometric mean of the expression of the previously ranked genes was calculated and then pair wise variations between sequential normalisation factors were calculated. Using the cut-off recommended by GeNorm of 0.15 the optimal number of reference genes for the data set in this study would be at least 5.

Mentions: In order to identify the genes that had the least variable expression, expression stability was evaluated using GeNorm and Normfinder software analysis. The pair wise variation between the normalisation factors calculated by GeNorm steadily decreased after inclusion of the fourth additional reference gene and falls below the cut-off of 0.15 that is suggested by the GeNorm programme after adding the fifth gene [27] (Figure 2). The ranking of the potential reference genes by GeNorm and Normfinder is given in Table 4.


Leukocyte count affects expression of reference genes in canine whole blood samples.

Piek CJ, Brinkhof B, Rothuizen J, Dekker A, Penning LC - BMC Res Notes (2011)

Pairwise variations between 2 sequential normalization factors including an increasing number of potential reference genes . To determine the optimal number of reference genes, first the geometric mean of the expression of the previously ranked genes was calculated and then pair wise variations between sequential normalisation factors were calculated. Using the cut-off recommended by GeNorm of 0.15 the optimal number of reference genes for the data set in this study would be at least 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045956&req=5

Figure 2: Pairwise variations between 2 sequential normalization factors including an increasing number of potential reference genes . To determine the optimal number of reference genes, first the geometric mean of the expression of the previously ranked genes was calculated and then pair wise variations between sequential normalisation factors were calculated. Using the cut-off recommended by GeNorm of 0.15 the optimal number of reference genes for the data set in this study would be at least 5.
Mentions: In order to identify the genes that had the least variable expression, expression stability was evaluated using GeNorm and Normfinder software analysis. The pair wise variation between the normalisation factors calculated by GeNorm steadily decreased after inclusion of the fourth additional reference gene and falls below the cut-off of 0.15 that is suggested by the GeNorm programme after adding the fifth gene [27] (Figure 2). The ranking of the potential reference genes by GeNorm and Normfinder is given in Table 4.

Bottom Line: The disease category and the white blood cell count significantly affected reference gene expression.The discrepancy between the ranking of reference genes in this study by Normfinder and Genorm can be explained by differences between the experimental groups such as "disease category" and "WBC count".This stresses the importance of assessing the expression stability of potential reference genes for gene experiments in canine whole blood anew for each specific experimental condition.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Sciences of Companion Animals, Utrecht, Utrecht University, PO Box 80154, 3508 TD Utrecht, The Netherlands. C.J.Piek@uu.nl.

ABSTRACT

Background: The dog is frequently used as a model for hematologic human diseases. In this study the suitability of nine potential reference genes for quantitative RT-PCR studies in canine whole blood was investigated.

Findings: The expression of these genes was measured in whole blood samples of 263 individual dogs, representing 73 different breeds and a group of 40 mixed breed dogs, categorized into healthy dogs and dogs with internal and hematological diseases, and dogs that underwent a surgical procedure. GeNorm analysis revealed that a combination of 5 to 6 of the most stably expressed genes constituted a stable normalizing factor. Evaluation of the expression revealed different ranking of reference genes in Normfinder and GeNorm. The disease category and the white blood cell count significantly affected reference gene expression.

Conclusions: The discrepancy between the ranking of reference genes in this study by Normfinder and Genorm can be explained by differences between the experimental groups such as "disease category" and "WBC count". This stresses the importance of assessing the expression stability of potential reference genes for gene experiments in canine whole blood anew for each specific experimental condition.

No MeSH data available.


Related in: MedlinePlus