Ampliscribe t7 flash transcription kit

To synthesize the double-stranded (ds) RNA of the TmSpz6 gene, primers containing the T7 promoter sequence at their 5′ ends were designed using SnapDragon-Long dsRNA design software (Table 1). The PCR was conducted using AccuPower^® Pfu PCR PreMix with the TmSpz6_Fw and TmSpz6_Rv primers (Table 1) under the following cycling conditions: an initial denaturation step at 94 °C for 2 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, and extension at 72 °C for 30 s, with a final extension step at 72 °C for 5 min. PCR products were purified using the AccuPrep PCR Purification Kit (Bioneer, Daejeon, Korea), and dsRNA was synthesized using the Ampliscribe^TM T7-Flash^TM Transcription Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer’s instructions. After synthesis, the dsRNA was purified by precipitation with 5 M ammonium acetate and 80% ethanol, and then it was quantified with an Epoch spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The dsRNA for enhanced green fluorescent protein (dsEGFP) was synthesized for use as a control and was stored at −20 °C until use.

The full-length cDNA clone and its replication-defective mutant (pJE04-1601S_p12 and pJE04-1601S_p12-GAA, respectively) were each linearized with NheI (New England Biolabs), and the RNA transcripts were synthesized with T7 RNA polymerase using AmpliScribe^TM T7-Flash^TM Transcription Kit (Epicentre Biotechnologies, Madison, WI, USA). After in vitro transcription, RNA transcripts of the cDNA clones were capped using a ScriptCap m7G Capping System (Epicentre Biotechnologies). The integrity and yield of the synthesized RNAs were determined by agarose gel electrophoresis. An aliquot (2.5 µg) of the capped RNA was transfected into confluent PLC/PRF/5 cells in a well of a six-well plate using the TransIT-mRNA transfection kit (Mirus Bio, Madison, WI, USA) in accordance with the manufacturer’s recommendations. Following incubation at 37 °C for two days, the cells were washed with PBS(-), and then the culture medium was replaced with 2 mL of growth medium, and the cells were incubated at 35.5 °C. Every other day, half of the culture medium (1 mL) was replaced with fresh growth medium. The collected culture medium was centrifuged at 1300× g at room temperature for 2 min, and the supernatants were stored at −80 °C until use.

The primers were designed based on the nucleotide sequences available in GenBank: ferritin2 (Fer2LCH) (XM_624073.4): forward 5′-ATTTTTGGCAACTGCCTCTG-3′, reverse 5′-ATTCTCGAACACGGTCTGCT-3′; ferritin1 (Fer1HCH) (XM_016916248.1): forward 5′-CCCCGTCGATTAAAGTACGA-3′, reverse 5′-GCATGTTCTCTTTCTTCTGTAGCA-3′; GFP: forward 5′-GAGATACCCAGATCAT-3′, reverse 5′-GATGATATTCACCACTT-3′. Primers were fused with the T7 promoter sequence. Total RNA was isolated from the trophocytes and oenocytes of three worker bees at 3 days after emergence using TRIzol (15596018; Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The complementary DNA (cDNA) was synthesized by using Superscript III First-Strand Synthesis System for RT-PCR (18080-051; Invitrogen, Carlsbad, CA, USA). The cDNA was transferred into E. coli by using Topo TA cloning Kit for sequencing (450030, Invitrogen, Carlsbad, CA, USA). The plasmid was isolated with QIAprep Spin Miniprep Kit (27104, Qiagen, Valencia, CA, USA). The double-stranded (dsDNA) was produced by PCR using the T7 primers and purified by QIA Quick Gel Extraction Kit (28704, Qiagen, Valencia, CA, USA). Finally, dsRNA was synthesized and purified by using AmpliScribe^TM T7-Flash^TM Transcription Kit (ASF3257, Epicentre Biotechnologies, Madison, WI, USA), and diluted with nuclease-free water to a final concentration of 5 μg/μl.