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

Cinar MU, Islam MA, Uddin MJ, Tholen E, Tesfaye D, Looft C, Schellander K - BMC Res Notes (2012)

Bottom Line: However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions.There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms.In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of the animals.

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: To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs) is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in pigs are relatively rare and have never been performed in porcine alveolar macrophages (AMs). In this study, expression stability of putative housekeeping genes were identified in the porcine AMs in response to the stimulation with two pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Three different algorithms (geNorm, Normfinder and BestKeeper) were applied to assess the stability of HKGs.

Results: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by qRT-PCR in AMs that were stimulated by LPS and LTA in vitro. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.0001). geNorm software revealed that SDHA, B2M and RPL4 showed a high expression stability in the irrespective to the stimulation group, while SDHA, YWHAZ and RPL4 showed high stability in non-stimulated control group. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that SDHA was the most stable gene in all the groups. 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.

Conclusions: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms. In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of the animals.

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Ranking of nine candidate reference genes using geNorm and NormFinder softwares. (a-e) geNorm ranks the candidate reference genes based on their stability parameter M. The lower the M value, the higher the expression stability. (f-j) NormFinder ranks the genes based on a calculated stability value. The lower the stability value, the higher the expression stability. Irrespective to stimulation: when all the stimulated and non-stimulated control were considered together; Control: no stimulation; 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-e) geNorm ranks the candidate reference genes based on their stability parameter M. The lower the M value, the higher the expression stability. (f-j) NormFinder ranks the genes based on a calculated stability value. The lower the stability value, the higher the expression stability. Irrespective to stimulation: when all the stimulated and non-stimulated control were considered together; Control: no stimulation; LPS: lipopolysaccharide; LTA: lipoteichoic acid; LPS + LTA (combined): lipopolysaccharide used together with lipoteichoic acid.

Mentions: Transcription profiling using qRT-PCR assays was then performed with these nine candidate genes, in samples from the four different conditions of AM cultures (LPS, LTA, combined LPS and LTA, and control). These raw Ct data were then analysed using different algorithms to identify the most suitable candidate genes. In each independent culture, the 9 genes were ranked according to their gene expression stability measure "M" (Figure 4a-e, 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) [8]. All genes presented an M value below 1.5, which is the default limit for acceptable expression stability as defined by Vandesompele et al. [8]. Figure 4a shows the ranking of the nine candidate reference genes across the AMs based on their stability values without considering the type of stimulation of cells i.e. irrespective of stimulation group. SDHA, B2M and RPL4 were identified as the most stable HKGs (Figure 4a) in the irrespective of stimulation group. In case of the control group, geNorm showed that SDHA, B2M and RPL4 were the most stable HKGs (Figure 4b). When AMs were stimulated with Gram negative bacterial product LPS, geNorm identified B2M, SDHA and YWHAZ as the most stable HKGs (Figure 4c). YWHAZ, PPIA and RPL4 were the most stably expressed HKGs in the case of Gram-positive bacterial product (LTA) stimulation group (Figure 4d). When LPS was used combined with LTA for the stimulation of AMs, HPRT1, YWHAZ and SDHA remained the most stable genes (Figure 4e). All investigated groups identified GAPDH as the least stable reference gene by geNorm (Figure 4a, c, d and 4e) except in control group where BLM was the least stable HKG (Figure 4b).


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

Cinar MU, Islam MA, Uddin MJ, Tholen E, Tesfaye D, Looft C, Schellander K - BMC Res Notes (2012)

Ranking of nine candidate reference genes using geNorm and NormFinder softwares. (a-e) geNorm ranks the candidate reference genes based on their stability parameter M. The lower the M value, the higher the expression stability. (f-j) NormFinder ranks the genes based on a calculated stability value. The lower the stability value, the higher the expression stability. Irrespective to stimulation: when all the stimulated and non-stimulated control were considered together; Control: no stimulation; 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=PMC3306271&req=5

Figure 4: Ranking of nine candidate reference genes using geNorm and NormFinder softwares. (a-e) geNorm ranks the candidate reference genes based on their stability parameter M. The lower the M value, the higher the expression stability. (f-j) NormFinder ranks the genes based on a calculated stability value. The lower the stability value, the higher the expression stability. Irrespective to stimulation: when all the stimulated and non-stimulated control were considered together; Control: no stimulation; LPS: lipopolysaccharide; LTA: lipoteichoic acid; LPS + LTA (combined): lipopolysaccharide used together with lipoteichoic acid.
Mentions: Transcription profiling using qRT-PCR assays was then performed with these nine candidate genes, in samples from the four different conditions of AM cultures (LPS, LTA, combined LPS and LTA, and control). These raw Ct data were then analysed using different algorithms to identify the most suitable candidate genes. In each independent culture, the 9 genes were ranked according to their gene expression stability measure "M" (Figure 4a-e, 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) [8]. All genes presented an M value below 1.5, which is the default limit for acceptable expression stability as defined by Vandesompele et al. [8]. Figure 4a shows the ranking of the nine candidate reference genes across the AMs based on their stability values without considering the type of stimulation of cells i.e. irrespective of stimulation group. SDHA, B2M and RPL4 were identified as the most stable HKGs (Figure 4a) in the irrespective of stimulation group. In case of the control group, geNorm showed that SDHA, B2M and RPL4 were the most stable HKGs (Figure 4b). When AMs were stimulated with Gram negative bacterial product LPS, geNorm identified B2M, SDHA and YWHAZ as the most stable HKGs (Figure 4c). YWHAZ, PPIA and RPL4 were the most stably expressed HKGs in the case of Gram-positive bacterial product (LTA) stimulation group (Figure 4d). When LPS was used combined with LTA for the stimulation of AMs, HPRT1, YWHAZ and SDHA remained the most stable genes (Figure 4e). All investigated groups identified GAPDH as the least stable reference gene by geNorm (Figure 4a, c, d and 4e) except in control group where BLM was the least stable HKG (Figure 4b).

Bottom Line: However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions.There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms.In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of the animals.

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: To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs) is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in pigs are relatively rare and have never been performed in porcine alveolar macrophages (AMs). In this study, expression stability of putative housekeeping genes were identified in the porcine AMs in response to the stimulation with two pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Three different algorithms (geNorm, Normfinder and BestKeeper) were applied to assess the stability of HKGs.

Results: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by qRT-PCR in AMs that were stimulated by LPS and LTA in vitro. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.0001). geNorm software revealed that SDHA, B2M and RPL4 showed a high expression stability in the irrespective to the stimulation group, while SDHA, YWHAZ and RPL4 showed high stability in non-stimulated control group. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that SDHA was the most stable gene in all the groups. 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.

Conclusions: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms. In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of the animals.

Show MeSH