Monarch rna mini prep kit

Total RNA was isolated from cells using the Monarch RNA Mini-Prep Kit (New England Biolabs, Ipswich, MA, USA). Small RNA fraction was prepared using the ‘Fractionation of Small and Large RNA’ protocol for the above kit.

For Tc-en qPCR experiments, embryos were injected with either 1 or 2 ug/uL Tc-en at 4 h AEL and collected at later developmental stages, roughly (24, 31, 48, 50 h AEL). Total RNA was isolated from 50 or more buffer-injected controls and dsRNA-injected eggs using Trizol (Invitrogen), according to the manufacturer’s instructions. The aqueous phase was purified using the NEB Monarch RNA mini-prep kit (NEB #T2010). 10 ng of RNA was used in each reaction of the NEB Luna Universal One-Step RT-qPCR kit (cat # E3005). RT-qPCR reactions were carried out in triplicate, and melting curves were examined to ensure single products. Results were quantified using the CFX Maestro software from Biorad using the ‘‘delta-delta Ct” method and normalized to Histone 3 (H3) transcript levels (Supplemental Figure S2). Primer sequences used are: H3 F: 5′-CTGCCCTTCCAGAGATTGGT-3′; H3 R: 5′-GAACAGACCCACGAGGTACG-3′; en F: 5′-CGCAGGGACTCTACAACCAC-3′; en R: 5′-CGAGATTTGCCTTCGCTCTC-3′; inv F: 5′-GCAAGCCGAAGAAGGTTGTG-3′; inv R: 5′-TTCTTGACTCGCCTGGTTCG-3′; hh F: 5′-CACTGAAGGACGCATCGGAA-3′; hh R: 5′-GGTTCATCACCGAAATCGCC-3′.

Tumors were extracted from euthanized fish and homogenized inside a 1.5 ml Eppendorf tube using a pestle connected to a Pellet Pestle Cordless Motor (DWK Life Sciences Kimble Kontes). Tumor RNA was extracted using NEB’s Monarch RNA mini prep kit. cDNA was synthesized from the extracted RNA using Invitrogen’s First Strand Synthesis cDNA kit. Following cDNA synthesis, real-time qPCR was performed in 384-well plates using Applied Biosystem’s Sybr Green qPCR MasterMix (Comparative Cт (ΔΔCт) method). Results were compiled and analyzed using the QuantStudio 7 Flex system (Applied Biosciences). PCR primers are provided in Supplementary file 4. All assays were completed using technical replicates, with at least three tumors tested per experimental group.