Identification of suitable reference genes for real-time RT-PCR normalization in Bidens laevis during pesticide and cold stress

LUKASZEWICZ, GERMÁN; AMÉ, MARÍA VALERIA; MENONE, MIRTA

Cracovia

Encuentro; 3rd Young Environmental Scientist Meeting.; 2013

SETAC

Environmental monitoring demands the usage of new species and sensitive methods in order to get accurate regional biomonitoring. The aquatic macrophyte Bidens laevis L. (fam.: Asteraceae) has desirable growing characteristics for use in laboratory assays, has proven to respond positively to genotoxic compounds and showed enzymatic response when exposed to the pesticide endosulfan. Nowadays, gene expression offers crucial information when analysing responses to xenobiotic compounds. It is well known that Real Time PCR (RT-qPCR) is the preferred method for studying gene expression because of its sensitivity, precision and robustness but it requires the use of reference genes (RG) with stable expression among different treatments, as well as different tissues and growth stages. The aim of this study was to analyse the expression of six candidate genes, Elongation Factor 1-alfa (EF1a), Beta-Actin (BACT), Histone 3 (H3), Nicotinamide Adenine Dinucleotide Dehydrogenase (NADHD) and glyceraldehyde-3-phosphate-dehydrogenase (GADPH),  in B. laevis in three tissues (root, stem and leaves) under four conditions: 1-control-: Large plants (>1500mg), temperature 22ºC, media: Hoagland solution; 2 -short-: Short plants (<250mg), temperature 22ºC, media: Hoagland solution; 3 -xenobiotic-: Large plants, temperature 22ºC, media: Hoagland solution + endosulfan 10ug/L; 4 -cold- Large plants, temperature 5ºC, media: Hoagland solution. All plants remained 24hs under each condition with 3 replicates tested for each one. The analysis of the stability of the proposed RG was performed with 4 different software methods (deltaCT, GeNorm, Bestkeeper, Normfinder) through the online software RefFinder. Primer specificity and amplification efficiency were verified for each gene. The overall analysis showed that NADHD and H3 are the most stable genes between different treatments and tissues. However, some exceptions arose when analysing specific tissues (BACT and NADHD were the most stable in leaves) and treatments (H3, NADHD and EF-1a showed the most stable expression among different tissues under one treatment).