Synthesis of dsRNA was performed using Ambion MEGAscript T7 Transcription Kit (ThermoFisher Scientific). dsRNAs for experiment and mock control treatment were synthesized using cDNA sequences from D. melanogaster CG7630 and human IGFBP2, respectively. The primers used for the amplification of T7 transcription template are listed in the table of primers (Table 1 ). For RNAi experiments, S2R+ cells were plated on 12‐well plates and treated for 1 h with 5 μg synthetic dsRNA in Schneider’s medium without serum. After treatment, serum was added and cells were harvested after 96 h.
Ambion megascript t7 transcription kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ambion MEGAscript T7 Transcription Kit is a laboratory product designed for in vitro transcription. It enables the efficient synthesis of high yields of RNA from DNA templates using the T7 RNA polymerase system.
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Lab products found in correlation
2 protocols using ambion megascript t7 transcription kit
1
Drosophila dsRNA Synthesis and RNAi
2
RNAi-Mediated Gene Silencing in Insects
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PCR was carried out using a gene-specific primer pair containing a T7 promoter sequence (5ʹ-TAATACGACTCACTATAGGG-3ʹ) at the 5′ end and a recombinant plasmid containing OcHsp70 as template. Thereafter, the PCR product was used as a template for dsRNA synthesis using Ambion™ MEGAscript® T7 Transcription Kit (Thermo-Fisher Scientific, CA, United States) according to the recommended protocol. The double-stranded green fluorescent protein (gfp) RNA, dsgfp, was used as blank (negative) control. Finally, the quality of dsOchsp70 was assessed using 1% agarose gel electrophoresis and quantified to 10 μg/ul. dsOcHsp70 and dsgfp were stored at −80 C for subsequent experiments (Jin et al., 2020).
For the RNAi experiment, newly emerged adults (males and females <12 h after eclosing) were injected with 500 ng of dsRNA in 100 nL water solution at the abdomen using the Nanoject III Programmable Nanoliter Injector (Drummond Scientific Co., Inc, PA, United States). At 5, 10, 15, and 20 d post injection (PI), the five injected adults of each biological replicate were collected for the evaluation of silencing efficiency using qPCR. The primers used in this study are listed inSupplementary Table S1 .
For the RNAi experiment, newly emerged adults (males and females <12 h after eclosing) were injected with 500 ng of dsRNA in 100 nL water solution at the abdomen using the Nanoject III Programmable Nanoliter Injector (Drummond Scientific Co., Inc, PA, United States). At 5, 10, 15, and 20 d post injection (PI), the five injected adults of each biological replicate were collected for the evaluation of silencing efficiency using qPCR. The primers used in this study are listed in
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