Genejet rna purification kit total rna purification protocol

RNA extractions were carried out according to the GeneJET RNA Purification Kit Total RNA Purification Protocol (Thermo Fisher Scientific Inc,, Waltham, MA, USA). RNA quality and quantity was analyzed spectrophotometrically using a BioDrop µLITE (BioDrop, Cambridge, UK, USA). The iScript cDNA Synthesis Kit for reverse transcription PCR (Bio-Rad Laboratories, Hercules, CA, USA) was used according to the manufacturer’s instructions using 360 ng of RNA and carried out in an Eppendorf Mastercycler Nexus Gradient thermocycler (Eppendorf, Hauppauge, NY, USA) according the reaction protocol provided by iScript.

To measure changes in gene expression, whole-embryo RNA was first isolated. Pools of 10–12 embryos were exposed to butylparaben as described above, fixed at 3 dpf (due to the significant pancreatic morphological variants observed at this time point; Figure 1 and 2, Table 1) in 100 µL RNA-Later (Thermo Fisher Scientific), and stored at −80 °C. Prior to RNA extraction, all materials and surfaces were sprayed with RNase Away® (Molecular BioProducts, Thermo Fisher Scientific). RNA extractions were carried out according to the GeneJET RNA Purification Kit Total RNA Purification Protocol (Thermo Fisher Scientific Inc., Waltham, MA). Samples were sonicated for one second at 15 % amplification with a Branson Digital Sonifier® (Danbury, CT) that was sterilized with 75 % ethanol between each sonication. RNA concentration and quality was analyzed using a BioDrop µLITE (BioDrop, Cambridge, UK). Reverse transcription to cDNA was carried out according to the manufacturer’s protocol using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) with an Eppendorf Mastercycler® nexus gradient thermal cycler (Eppendorf, Hauppague, NY) according to the manufacturer’s protocol. A total of 5 biological replicates per exposure concentration were collected from three independent experiments.