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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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Average cycle threshold (Cq) values of candidate reference genes tested in PBMCs under different conditions. The values are the average RT-qPCR cycle threshold numbers (Cq values). The box plot indicates sample’s range, median, normality of the distribution, and skew of the distribution. Letters indicate a significant difference in average Cq value. Average Cq values that have the same letter are not significantly different (P > 0.05).
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Figure 2: Average cycle threshold (Cq) values of candidate reference genes tested in PBMCs under different conditions. The values are the average RT-qPCR cycle threshold numbers (Cq values). The box plot indicates sample’s range, median, normality of the distribution, and skew of the distribution. Letters indicate a significant difference in average Cq value. Average Cq values that have the same letter are not significantly different (P > 0.05).

Mentions: Transcript abundance of commonly used reference genes were analysed in the different samples by direct comparison of their cycle threshold (Cq), assuming equal Cq for equal transcript number since all RT-qPCR reactions were performed with an equal quantity of total RNA. Figure 2 showed that nine selected genes presented mean Cq-values that ranged from 27.78 to 33.04 cycles. Lowest and highest Cq-values were observed for PPIA (Cq 19.15) and TBP (Cq 37.62), respectively (Figure 2). Cq value of the selected genes showed a reasonable dispersion to moderately low expression levels (Figure 2). The highest variation was observed in GAPDH (min Cq 20.38 – max Cq 37.00) and followed by RPL4 (min Cq 19.44 – max Cq 35.74). BLM showed the lowest dispersion (min Cq 26.05 – max Cq 37.03) and mRNA expression as indicated by Cq-value around 33 cycles over the stimulations and culture conditions indicated by narrow whiskers of the box (Figure 2). SDHA was the second lowest varied (min Cq 25.09 – max Cq 36.65) and expressed gene (mean Cq 32.57) in our study (Figure 2). PPIA (mean Cq 27.78) was found to be highest expressed (mean Cq 27.78) gene among stimulations and different culture conditions (Figure 2). This followed by the expression of B2M gene (mean Cq 28.74). According to variance analysis, the expressions of eight genes were different from each other (P < 0.05) (Figure 2).


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)

Average cycle threshold (Cq) values of candidate reference genes tested in PBMCs under different conditions. The values are the average RT-qPCR cycle threshold numbers (Cq values). The box plot indicates sample’s range, median, normality of the distribution, and skew of the distribution. Letters indicate a significant difference in average Cq value. Average Cq values that have the same letter are not significantly different (P > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Average cycle threshold (Cq) values of candidate reference genes tested in PBMCs under different conditions. The values are the average RT-qPCR cycle threshold numbers (Cq values). The box plot indicates sample’s range, median, normality of the distribution, and skew of the distribution. Letters indicate a significant difference in average Cq value. Average Cq values that have the same letter are not significantly different (P > 0.05).
Mentions: Transcript abundance of commonly used reference genes were analysed in the different samples by direct comparison of their cycle threshold (Cq), assuming equal Cq for equal transcript number since all RT-qPCR reactions were performed with an equal quantity of total RNA. Figure 2 showed that nine selected genes presented mean Cq-values that ranged from 27.78 to 33.04 cycles. Lowest and highest Cq-values were observed for PPIA (Cq 19.15) and TBP (Cq 37.62), respectively (Figure 2). Cq value of the selected genes showed a reasonable dispersion to moderately low expression levels (Figure 2). The highest variation was observed in GAPDH (min Cq 20.38 – max Cq 37.00) and followed by RPL4 (min Cq 19.44 – max Cq 35.74). BLM showed the lowest dispersion (min Cq 26.05 – max Cq 37.03) and mRNA expression as indicated by Cq-value around 33 cycles over the stimulations and culture conditions indicated by narrow whiskers of the box (Figure 2). SDHA was the second lowest varied (min Cq 25.09 – max Cq 36.65) and expressed gene (mean Cq 32.57) in our study (Figure 2). PPIA (mean Cq 27.78) was found to be highest expressed (mean Cq 27.78) gene among stimulations and different culture conditions (Figure 2). This followed by the expression of B2M gene (mean Cq 28.74). According to variance analysis, the expressions of eight genes were different from each other (P < 0.05) (Figure 2).

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