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Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA.

Cinar MU, Islam MA, Pröll M, Kocamis H, Tholen E, Tesfaye D, Looft C, Schellander K, Uddin MJ - BMC Res Notes (2013)

Bottom Line: The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study.PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured.In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Animal Sciences, Unit of Animal Breeding and Husbandry, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany.

ABSTRACT

Background: As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs.

Results: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.

Conclusion: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

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Expression levels of a representative subset of nine reference genes. a) B2M, b) BLM, c) GAPDH, d) HPRT1, e) PPIA, f) RPL4, g) SDHA, h) TBP and i) YWHAZ. NC (not-culture): cells which were not used for in vitro culture (in this case, PBMCs isolated from blood was used); Control: cells which were used for in vitro culture but was not stimulated; LPS: lipopolysaccharide; LTA: lipoteichoic acid; combined (LPS + LTA): lipopolysaccharide used together with lipoteichoic acid. Letters indicate a significant difference in average Cq value (P < 0.05).
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Figure 3: Expression levels of a representative subset of nine reference genes. a) B2M, b) BLM, c) GAPDH, d) HPRT1, e) PPIA, f) RPL4, g) SDHA, h) TBP and i) YWHAZ. NC (not-culture): cells which were not used for in vitro culture (in this case, PBMCs isolated from blood was used); Control: cells which were used for in vitro culture but was not stimulated; LPS: lipopolysaccharide; LTA: lipoteichoic acid; combined (LPS + LTA): lipopolysaccharide used together with lipoteichoic acid. Letters indicate a significant difference in average Cq value (P < 0.05).

Mentions: PBMC mRNA expression differences were investigated in nine reference genes in no cultured, cultured with no stimulation, or stimulated with LPS, LTA or both. There were some fluctuations in the expression level of these genes in certain conditions. The expression differences of these genes are shown in Figure 3. The variance analysis results between treatment groups and time of stimuli to the PBMCs are shown in supplementary Table 1. Cell harvest time significantly affected the expression level of reference genes (P ≤ 0.02) (Supplementary Table 1). In all genes except SDHA, no statistical difference (P > 0.05) was observed between no culture (NC) and no stimulation (control) group (Figure 3). In the SDHA, no stimulation control group showed lower Cq value compared to no culture group (P < 0.05) (Figure 3g). When no culture group and no stimulation control group were compared with the stimulated groups, the expression levels of all genes were decreased in stimulated groups (Figure 3, Table 1). Within the stimulated groups, expression of BLM, GAPDH, HPRT1, SDHA, and YWHAZ was not effected from stimulation type (LPS, LTA or combined) (Figure 3). With LPS or LTA stimulation, mRNA expression levels of nine genes were decreased compared to control group (Figure 3a-i).


Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA.

Cinar MU, Islam MA, Pröll M, Kocamis H, Tholen E, Tesfaye D, Looft C, Schellander K, Uddin MJ - BMC Res Notes (2013)

Expression levels of a representative subset of nine reference genes. a) B2M, b) BLM, c) GAPDH, d) HPRT1, e) PPIA, f) RPL4, g) SDHA, h) TBP and i) YWHAZ. NC (not-culture): cells which were not used for in vitro culture (in this case, PBMCs isolated from blood was used); Control: cells which were used for in vitro culture but was not stimulated; LPS: lipopolysaccharide; LTA: lipoteichoic acid; combined (LPS + LTA): lipopolysaccharide used together with lipoteichoic acid. Letters indicate a significant difference in average Cq value (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3584940&req=5

Figure 3: Expression levels of a representative subset of nine reference genes. a) B2M, b) BLM, c) GAPDH, d) HPRT1, e) PPIA, f) RPL4, g) SDHA, h) TBP and i) YWHAZ. NC (not-culture): cells which were not used for in vitro culture (in this case, PBMCs isolated from blood was used); Control: cells which were used for in vitro culture but was not stimulated; LPS: lipopolysaccharide; LTA: lipoteichoic acid; combined (LPS + LTA): lipopolysaccharide used together with lipoteichoic acid. Letters indicate a significant difference in average Cq value (P < 0.05).
Mentions: PBMC mRNA expression differences were investigated in nine reference genes in no cultured, cultured with no stimulation, or stimulated with LPS, LTA or both. There were some fluctuations in the expression level of these genes in certain conditions. The expression differences of these genes are shown in Figure 3. The variance analysis results between treatment groups and time of stimuli to the PBMCs are shown in supplementary Table 1. Cell harvest time significantly affected the expression level of reference genes (P ≤ 0.02) (Supplementary Table 1). In all genes except SDHA, no statistical difference (P > 0.05) was observed between no culture (NC) and no stimulation (control) group (Figure 3). In the SDHA, no stimulation control group showed lower Cq value compared to no culture group (P < 0.05) (Figure 3g). When no culture group and no stimulation control group were compared with the stimulated groups, the expression levels of all genes were decreased in stimulated groups (Figure 3, Table 1). Within the stimulated groups, expression of BLM, GAPDH, HPRT1, SDHA, and YWHAZ was not effected from stimulation type (LPS, LTA or combined) (Figure 3). With LPS or LTA stimulation, mRNA expression levels of nine genes were decreased compared to control group (Figure 3a-i).

Bottom Line: The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study.PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured.In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Animal Sciences, Unit of Animal Breeding and Husbandry, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany.

ABSTRACT

Background: As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs.

Results: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.

Conclusion: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

Show MeSH
Related in: MedlinePlus