mRNA-Seq of Myriophyllum spicatum exposed to low-effect concentrations of the herbicide bentazone and the pharmaceutical atorvastatin against untreated control groups
mRNA-Seq of Myriophyllum spicatum exposed to low-effect concentrations of the herbicide bentazone and the pharmaceutical atorvastatin against untreated control groups
25 to 35 mg of fresh shoot tip tissues were collected from the test vessels. The tissue was homogenized with a lysing matrix (¼ Ceramic Sphere, MP Biomedicals) in a FastPrep-24© 5G (MP Biomedicals) at 5 m/s for 60 s at room temperature. The lysis was performed in buffer PH from the RapidPure RNA Plant Kit (MP Biomedicals). A preculture was prepared a week prior to test start in Smart & Barko medium with plants from a sterile stock culture under test conditions according to the OECD guideline test No. 239 in Smart & Barko medium. Plants were grown at a light intensity of 120 to 160 µE∙m-2∙s-1 at the water surface and a 16 h light/8 h dark cycle with randomized placements at a temperature of 20±2 °C. Each shoot, with a length of 6±1 cm, was planted in 100 g of sediment in a 50 mL beaker with 3 cm of the shoot above the surface. Bentazone (CAS 25057-89-0) was purchased from Sigma-Aldrich (St. Louis, USA), atorvastatin calcium salt (CAS 134523-03-8) was purchased from abcr (Karlsruhe, Germany). Low effect concentrations corresponding to the EC5 and EC20 (ECx indicating x% inhibition of growth) were determined in preliminary tests were prepared in Smart & Barko medium at a pH of 7.5 to 8. 500 mL measuring cylinders, as test vessels, were filled with approximately 600 mL of test solution and analogously 600 mL of pure Smart & Barko medium as a control. Three plants per exposure condition were transferred from the pre-culture into their respective test vessels and incubated for a total of three days under random arrangement under conditions as indicated in the growth protocol. The total RNA was extracted with the RapidPure RNA Plant Kit (MP Biomedicals) according to the manufacturer's instruction (Application Manual Revision 112722050-14APR) following a three minute incubation on ice after lysis. The quantity and purity of RNA samples was assessed via spectrometric measurements with a NanoDrop2000 (Thermo Scientific) and the RNA integrity number (RIN) determined with a Bioanalyzer 2100 system (Agilent Technologies). Samples were stored at -80°C prior to sequencing. High-quality RNA extracts with RINs > 8 were used to prepary cDNA libraries using poly(A) RNA selection with the TruSeq RNA Library Prep Kit v2 (Illumina) according to a strand specific protocol. Libraries were assessed with a Fragment Analyzer system (Agilent) prior to sequencing.