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Nanoparticles Affect the Expression Stability of Housekeeping Genes in Plant Cells.

ABSTRACT: Purpose:We report on the expression stability of several housekeeping/reference genes that can be used in the normalization of target gene expression in quantitative real-time PCR (qRT-PCR) analysis of plant cells challenged with metal nanoparticles (NPs). Materials and Methods:Uniform cell suspension cultures of Hypericum perforatum were treated with 25 mg/l silver and gold NPs (14-15 nm in diameter). Cells were collected after 0.5, 4.0, and 12 h. The total RNA isolated from the cells was analyzed for the stability of ACT2, ACT3, ACT7, EF1-?, GAPDH, H2A, TUB-?, TUB-?, and 18S rRNA genes using qRT-PCR. The cycle threshold (Ct) values of the genes were analyzed using the geNorm, NormFinder, BestKeeper, and RefFinder statistical algorithms to rank gene stability. The stability of the top-ranked genes was validated by normalizing the expression of HYP1. Results:The expression of the tested housekeeping genes varied with treatment duration and NP types. EF1-? in gold NP treatment and TUB-? and EF1-? in silver NP treatment ranked among the top three positions. However, none of the genes retained their top ranking with time and across NP types. Conclusion:EF1-? can be used as a reference for treatment involving both silver and gold NPs in H. perforatum cells. TUB-? can be used only for silver NP-treated cells. The expression instability of most of the housekeeping genes highlights the importance of systematic standardization of reference genes for NP treatment conditions to draw proper conclusions on the target gene expression.

SUBMITTER: Selvakesavan RK

PROVIDER: S-EPMC7431599 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Nanoparticles Affect the Expression Stability of Housekeeping Genes in Plant Cells.

Selvakesavan Rajendran K RK Franklin Gregory G

Nanotechnology, science and applications 20200813

<h4>Purpose</h4>We report on the expression stability of several housekeeping/reference genes that can be used in the normalization of target gene expression in quantitative real-time PCR (qRT-PCR) analysis of plant cells challenged with metal nanoparticles (NPs).<h4>Materials and methods</h4>Uniform cell suspension cultures of <i>Hypericum perforatum</i> were treated with 25 mg/l silver and gold NPs (14-15 nm in diameter). Cells were collected after 0.5, 4.0, and 12 h. The total RNA isolated fr ...[more]

PMID: 32884247

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Project description:BACKGROUND: Gene expression analysis using real-time RT-PCR (qRT-PCR) is increasingly important in biological research due to the high-throughput and accuracy of qRT-PCR. For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes or internal control genes is required. The stability of reference genes has a tremendous effect on the results of relative quantification of gene expression by qRT-PCR. The expression stability of reference genes could vary according to tissues, age of individuals and experimental conditions. In the pig however, very little information is available on the expression stability of reference genes. The aim of this research was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in varieties of porcine tissues at different ages. RESULTS: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined in varieties of tissues collected from newborn, young and adult pigs. geNorm, NormFinder and BestKeeper software were used to rank the genes according to their stability. geNorm software revealed that RPL4, PPIA and YWHAZ showed high stability in newborn and adult pigs, while B2M, YWHAZ and SDHA showed high stability in young pigs. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that TBP was the most stable gene in newborn and young pigs, while PPIA was most stable in adult pigs. Moreover, geNorm software suggested that the geometric mean of three most stable gene would be the suitable combination for accurate normalization of gene expression study. CONCLUSIONS: Although, there was discrepancy in the ranking order of reference genes obtained by different analysing software methods, the geometric mean of the RPL4, PPIA and YWHAZ seems to be the most appropriate combination of housekeeping genes for accurate normalization of gene expression data in different porcine tissues at different ages.

Dataset Information

Nanoparticles Affect the Expression Stability of Housekeeping Genes in Plant Cells.

Publications

Nanoparticles Affect the Expression Stability of Housekeeping Genes in Plant Cells.

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