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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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Ranking of nine candidate reference genes using geNorm and NormFinder softwares. (a-g) GeNorm ranks the candidate reference genes based on their stability parameter M. The lower the M value, the higher the expression stability. (h-n) NormFinder ranks the genes based on a calculated stability value. The lower the stability value, the higher the expression stability. All: when all types of stimulated + control + no-culture were considered together. Irrespective to stimulation: when all the stimulated and non-stimulated control groups were considered together; Control: no stimulation; NC (no culture): PBMCs did not culture, just after isolation from blood, it was used for RNA isolation in order to perform mRNA expression study. LPS: lipopolysaccharide; LTA: lipoteichoic acid; LPS + LTA (combined): lipopolysaccharide used together with lipoteichoic acid.
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Figure 4: Ranking of nine candidate reference genes using geNorm and NormFinder softwares. (a-g) GeNorm ranks the candidate reference genes based on their stability parameter M. The lower the M value, the higher the expression stability. (h-n) NormFinder ranks the genes based on a calculated stability value. The lower the stability value, the higher the expression stability. All: when all types of stimulated + control + no-culture were considered together. Irrespective to stimulation: when all the stimulated and non-stimulated control groups were considered together; Control: no stimulation; NC (no culture): PBMCs did not culture, just after isolation from blood, it was used for RNA isolation in order to perform mRNA expression study. LPS: lipopolysaccharide; LTA: lipoteichoic acid; LPS + LTA (combined): lipopolysaccharide used together with lipoteichoic acid.

Mentions: Transcription profiling using RT-qPCR assays was then performed with these nine candidate genes, in samples from the five different conditions of AM cultures (LPS, LTA, combined LPS and LTA, control and no culture). These raw Cq data were then analysed using different algorithms to identify the most suitable candidate genes. In each independent culture, the nine genes were ranked according to their gene expression stability measure “M” (Figure 4a-g, left panel) with using the geNorm algorithm. Stepwise exclusion of the least stable gene allowed the genes to be ranked according to their M value (the lower the M value, the higher the gene’s expression stability) [9]. The ranking of the candidate reference genes for all 5 different cases was shown in Figure 4a-g. According to M values, RPL4, PPIA and B2M were the most stable reference genes across the PBMCs based on their stability values without considering the type of stimulation of cells i.e. irrespective of stimulation group (Figure 4a). In case of the control group, geNorm showed that PPIA, BLM and GAPDH were the most stable reference genes (Figure 4b). When PBMCs were stimulated with Gram-negative bacterial product LPS, geNorm identified PPIA, B2M and RPL4 as the most stable reference genes (Figure 4c). YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of Gram-positive bacterial product (LTA) stimulation group (Figure 4d). When LPS was used combined with LTA for the stimulation of PBMCs, YWHAZ, B2M and SDHA remained the most stable genes (Figure 4e). Figure 4f shows the ranking of the nine candidate genes for PBMCs under no culture condition, where PPIA, BLM and GAPDH were found to be most stably expressed reference genes. Among all investigated groups, in four groups, TBP was found to be the least stable reference gene by geNorm (Figure 4a, 4c, 4d, 4g) except in control, LPS and LTA combined and in the no culture group; where YWHAZ was the least stably expressed gene in the control and no culture group (Figure 4b and 4f) and GAPDH was found to be the least stably expressed gene in the LPS and LTA combined group (Figure 4e).


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)

Ranking of nine candidate reference genes using geNorm and NormFinder softwares. (a-g) GeNorm ranks the candidate reference genes based on their stability parameter M. The lower the M value, the higher the expression stability. (h-n) NormFinder ranks the genes based on a calculated stability value. The lower the stability value, the higher the expression stability. All: when all types of stimulated + control + no-culture were considered together. Irrespective to stimulation: when all the stimulated and non-stimulated control groups were considered together; Control: no stimulation; NC (no culture): PBMCs did not culture, just after isolation from blood, it was used for RNA isolation in order to perform mRNA expression study. LPS: lipopolysaccharide; LTA: lipoteichoic acid; LPS + LTA (combined): lipopolysaccharide used together with lipoteichoic acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Ranking of nine candidate reference genes using geNorm and NormFinder softwares. (a-g) GeNorm ranks the candidate reference genes based on their stability parameter M. The lower the M value, the higher the expression stability. (h-n) NormFinder ranks the genes based on a calculated stability value. The lower the stability value, the higher the expression stability. All: when all types of stimulated + control + no-culture were considered together. Irrespective to stimulation: when all the stimulated and non-stimulated control groups were considered together; Control: no stimulation; NC (no culture): PBMCs did not culture, just after isolation from blood, it was used for RNA isolation in order to perform mRNA expression study. LPS: lipopolysaccharide; LTA: lipoteichoic acid; LPS + LTA (combined): lipopolysaccharide used together with lipoteichoic acid.
Mentions: Transcription profiling using RT-qPCR assays was then performed with these nine candidate genes, in samples from the five different conditions of AM cultures (LPS, LTA, combined LPS and LTA, control and no culture). These raw Cq data were then analysed using different algorithms to identify the most suitable candidate genes. In each independent culture, the nine genes were ranked according to their gene expression stability measure “M” (Figure 4a-g, left panel) with using the geNorm algorithm. Stepwise exclusion of the least stable gene allowed the genes to be ranked according to their M value (the lower the M value, the higher the gene’s expression stability) [9]. The ranking of the candidate reference genes for all 5 different cases was shown in Figure 4a-g. According to M values, RPL4, PPIA and B2M were the most stable reference genes across the PBMCs based on their stability values without considering the type of stimulation of cells i.e. irrespective of stimulation group (Figure 4a). In case of the control group, geNorm showed that PPIA, BLM and GAPDH were the most stable reference genes (Figure 4b). When PBMCs were stimulated with Gram-negative bacterial product LPS, geNorm identified PPIA, B2M and RPL4 as the most stable reference genes (Figure 4c). YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of Gram-positive bacterial product (LTA) stimulation group (Figure 4d). When LPS was used combined with LTA for the stimulation of PBMCs, YWHAZ, B2M and SDHA remained the most stable genes (Figure 4e). Figure 4f shows the ranking of the nine candidate genes for PBMCs under no culture condition, where PPIA, BLM and GAPDH were found to be most stably expressed reference genes. Among all investigated groups, in four groups, TBP was found to be the least stable reference gene by geNorm (Figure 4a, 4c, 4d, 4g) except in control, LPS and LTA combined and in the no culture group; where YWHAZ was the least stably expressed gene in the control and no culture group (Figure 4b and 4f) and GAPDH was found to be the least stably expressed gene in the LPS and LTA combined group (Figure 4e).

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