Reliaprep rna miniprep system kit

Samples were collected at 1, 2, 3, 4, 5, and 30 dpf and at 1-year-old for adults. Whole zebrafish embryos and larvae or micro-dissected tissue from eyes, head, and tails were pooled and RNA was extracted using the ReliaPrep RNA Miniprep System kit (Promega, Madison, WI). RNA was treated with DNase I (New England Biolabs) and complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis kit (Bio-Rad). 600 ng of cDNA per sample were analyzed via qPCR using the SsoFast EvaGreen Supermix (Bio-Rad) or Power UP SYBR Green Master Mix (Thermo Fisher). All reactions were run with 3 technical replicates and repeated on at least 3 biological replicates for 40 cycles on a QuantStudio 3 RealTime PCR system and recorded with QuantStudio Design and Analysis software. Custom primers were designed for inpp5ka (ex9_F: 5′-TGGGACTGGATTGGGTTAT-3′; ex10_R:5′-GCTCCTCATTGAAAGACACC-3′) and inpp5kb (ex2_F: 5′-CGACCACTGACCTCTATGTG-3′; ex3_R: 5′-ATGAGGAGGTGACTCCATGT-3′) and housekeeping controls elongation factor 1 alpha eef1a (eef1a_F: 5′GGGCAAGGGCTCCTTCAA-3′; eef1a_R: 5′-CGCTCGGCCTTCAGTTTG-3′) and riboprotein L18 rpl18 (rpl18_F: 5′-GGCTAAGGTGATGTTTTCGTG-3′; rpl18_R: 5′-GCACATTGCCAATGTTCAGC-3′).

Total RNA of the control and exposed rats was isolated using from approximately 50 mg of frozen tissue according to TRIzol^TM manufacturer’s protocol (Invitrogen™,Carlsbad, CA, USA). Samples were homogenized in TRIzol^TM (50 mg/mL) with a T25 Ultra-turrax Digital High-Speed Homogenizer (IKA^®, Staufen, Germany). Extracted RNA was purified according to ReliaPrep™ RNA Miniprep System kit (Promega, Madison, WI, USA). The purity and quantity of RNA were evaluated spectrophotometrically using a NanoDrop™ 2000 (Thermo Scientific™, Madrid, Spain), showing concentrations between 370 and 2359 ng/μL and appropriate 260/280 nm and 260/230 nm ratios both around 2 (Table 1). RNA samples were stored until their dilution to 100 ng/μL with ultrapure Milli QH₂O system until their reverse transcription to cDNA.

The standards of standard solution stock (purity: 99%) of ENA (mw: 681.92 g/mol), ENB (mw: 639.82 g/mol), ENA1 (mw: 667.87 g/mol), ENB1 (mw: 653.85 g/mol) and phosphate buffer saline (PBS) were obtained from Sigma-Aldrich (Madrid, Spain). All the stock solutions were prepared by dissolving 1 mg of mycotoxin in 1 mL of pure methanol, obtaining a 1 mg/mL (1000 mg/L) solution. These stock solutions were diluted with methanol in order to obtain the appropriate multi-compounds working standard solutions. All the standards were kept at −20 °C.
For RNA extraction, TRIzol^TM reagent was purchased from Invitrogen™ (Carlsbad, CA, USA), whereas for its purification, the ReliaPrep™ RNA Miniprep System kit from Promega (Madison, WI, USA) was employed. Deionized water (resistivity < 18 MV cm) was obtained using a Milli-Q water purification system (Millipore, Bedford, MA, USA). TaqMan™ Reverse Transcription kit and PowerUp™ SYBR™ Green for quantitative Real-Time PCR (qPCR) analysis were purchased from Applied Biosystems (Carlsbad, CA, USA).