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• W151110305 abstract "BioTechniquesVol. 37, No. 6 BenchmarksOpen AccessInsulin stimulates double-stranded RNA uptake in Drosophila S2 cellsJohn C. March & William E. BentleyJohn C. MarchUniversity of Maryland, College Park, MD, USASearch for more papers by this author & William E. Bentley*Address correspondence to: William E. Bentley, Center for Biosystems Research, University of Maryland Biotechnology Institute, and Department of Chemical Engineering, University of Maryland, 5115 Plant Sciences Building, College Park, MD 20742, USA. e-mail: E-mail Address: bentley@eng.umd.eduUniversity of Maryland, College Park, MD, USASearch for more papers by this authorPublished Online:6 Jun 2018https://doi.org/10.2144/04376BM02AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail The advent of RNA interference (RNAi) has facilitated a boom in biochemical pathway analysis and functional genomics. RNAi refers to any RNA molecule that interferes with the expression of its homologous gene product (1–5). Also referred to as posttranscriptional gene silencing, RNAi is exquisitely specific for the targeted gene and encompasses sense, antisense, or double-stranded RNA (dsRNA) molecules, although it is commonly attributed with dsRNA as single-stranded RNA (ssRNA) effects have been traced to low levels of contaminating dsRNA (3,4).dsRNA of 220–700 bp has been shown to significantly reduce levels of messenger RNA (mRNA) transcript in Drosophila cell culture for a wide range of genes including insulin signaling pathway components (6), recombinant green fluorescent protein (GFP) (7), and for 91% of the genes associated with proliferation and survival (8). dsRNA of 500–700 bp can be transfected into Drosophila Schneider 2 (S2) cells by incubating them with fetal bovine serum (FBS) following serum starvation (6). However, FBS has numerous stimulatory effects and can greatly complicate metabolic studies, due its poorly characterized and variant composition. Recent work has taken advantage of serum-free media (SFM) for metabolic studies in cell culture (9,10). Unfortunately, while S2 cells can grow without serum, we were unable to stimulate dsRNA uptake utilizing only serum-free media (data not shown).In this study, we were able to remove FBS completely from dsRNA transfection by replacing it with bovine insulin. To test the effect of trying to replace FBS with insulin, we selected Cyclin E as a target gene for silencing. To test the effect of varying the amount of dsRNA used for silencing, we selected the tuberous sclerosis complex gene (TSC1) as a target. S2 cells were grown in Drosophila SFM (Invitrogen, Carlsbad, CA, USA). dsRNA was synthesized following a modified version of the method developed by Clemens and coworkers (6). Briefly, S2 cells were grown to 5 × 106 cells/mL, and RNA was extracted using an RNAqueous® kit (Ambion, Houston, TX, USA) according to the manufacturer's protocol, including incubation with DNase to remove any contaminatng DNA. First-strand templates of Cyclin E DNA were synthesized from the total mRNA using oligo(dT) primers and a RETROscript™ kit (Ambion) as per the manufacturer's instructions. Oligo(dT) primers were selected to further minimize any effects from contaminating DNA. A 630-bp region of first-strand DNA template was PCR-amplified using Cyclin E-specific primers (5′-ATGGGTTTAAATGCCAAGAGTGTTTGTTC-3′; 3′-CACCACCACTGGCGTCTGCTTGCTTCCACG-5′) or a 700-bp region with TSC1-specific primers (5′-ATGACGCTGGAGA-ACGAGGAGGCCAAGCGC-3′; 3′-CCATCTCCTTCCATCGCG-TATTGTTTACC-5′). T7 sequences (5′-TAATACGACTCACTATAGGGA-3′) were added to each template using PCR, making T7 templates. To transcribe ssRNA, T7 templates were used with the MEGAscript™ kit (Ambion) as per the manufacturer's instructions. The ssRNA synthesized was extracted using phenol/chloroform and resuspended in nuclease-free water to a concentration of approximately 3.3 µg/µL. ssRNA was incubated at 65°C for 30 min before being allowed to cool to room temperature on the benchtop. Subsequent to this annealing step, dsRNA was checked for size and integrity using agarose gel electrophoresis.Cells were seeded in triplicate to 1 × 106 cells/well in 0.7 mL SFM in 12-well plates and incubated with 15 µg/mL dsRNA against cyclin E, 15 µg/mL dsRNA against chloramphenicol acetyl transferase (CAT) (as a nonspecific control), or an equal volume of nuclease-free water. To determine the minimum amount of dsRNA needed for gene silencing and to demonstrate the generality of insulin-mediated uptake, cells were again seeded in triplicate to 1 × 106 cells/well in 0.7 mL SFM in 12-well plates and incubated with 6, 15, or 30 µg/mL dsRNA against TSC1 or an equal volume of nuclease-free water. dsRNA (15 µg/mL) against DsRed served as a nonspecific control. CAT and DsRed were used as controls because they have no significant similarity to any genes in the Drosophila genome [Basic Local Alignment Search Tool (BLAST) search]. The CAT and DsRed dsRNA fragments were 630 and 689 bp long, respectively. After 1 h incubation, SFM containing either 10% FBS (Sigma, St. Louis, MO, USA) or 1.75 µM bovine insulin (Sigma) was added to a final volume of 2.1 mL/well. The amount of insulin used was determined to be the optimum for growth in S2 cell culture with SFM. Higher amounts were tried, but resulted in cell lysis as determined by trypan blue dye staining (data not shown). The plates were incubated for 60 h at 27°C. RNA was extracted from the cells, and reverse transcription PCR (RT-PCR) was performed as described above for a 630-bp segment of Cyclin E or a 700-bp segment of TSC1 and an 866-bp segment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Each treatment's triplicate RNA samples were pooled before reverse transcription. Results (Figure 1) indicate that insulin worked as well or better than FBS at facilitating posttranscriptional gene silencing in S2 cells and that the amount dsRNA needed for silencing using insulin has to be determined for each gene under study.Figure 1. Insulin stimulates uptake of double-stranded RNA (dsRNA) in S2 cells.(A) Reverse transcription PCR (RT-PCR) of total RNA from S2 cells using oligo(dT) primers for reverse transcription and Cyclin E-specific primers for PCR. Conditions are listed across the top with cells either receiving dsRNA treatment against Cyclin E (dsCycE), chloramphenicol acetyl transferase (dsCAT), or no dsRNA treatment (Nothing). Either insulin or fetal bovine serum (FBS) was used to stimulate dsRNA uptake. (B) Reverse transcripts were also subject to PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers as a loading control. (C) RT-PCR of total RNA from S2 cells using oligo(dT) primers for reverse transcription, and TSC1-specific primers for PCR following treatment with nuclease-free water and no insulin (No Ins), nuclease-free water and 1.75 µM insulin stimulation (No dsRNA), 6 µg/mL dsRNA against TSC1 (6-TSC1), 15 µg/mL dsRNA against TSC1 (15-TSC1), 30 µg/mL dsRNA against TSC1, and 15 µg/mL dsRNA against DsRed (15-DsRed). (D) Reverse transcripts were also subject to PCR using GAPDH primers as a loading control. PCR products are aligned with the appropriate treatment.AcknowledgmentsThis work was funded by National Institutes of Health (NIH) grant no. 1R01GM70851-01.Competing Interests StatementThe authors declare no conflicts of interest.References1. Fire, A., S.Q. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811.Crossref, Medline, CAS, Google Scholar2. Montgomery, M.K. and A. Fire. 1998. Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression. Trends Genet. 14:255–258.Crossref, Medline, CAS, Google Scholar3. Montgomery, M.K., S. Xu, and A. Fire. 1998. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 95:15502–15507.Crossref, Medline, CAS, Google Scholar4. Rocheleau, C.E., W.D. Downs, R.L. Lin, C. Wittmann, Y.X. Bei, Y.H. Cha, M. Ali, J.R. Priess, and C.C. Mello. 1997. Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos. Cell 90:707–716.Crossref, CAS, Google Scholar5. Timmons, L. and A. Fire. 1998. Specific interference by ingested dsRNA. Nature 395:854–854.Crossref, Medline, CAS, Google Scholar6. Clemens, J.C., C.A. Worby, N. Simonson-Leff, M. Muda, T. Maehama, B.A. Hemmings, and J.E. Dixon. 2000. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97:6499–6503.Crossref, Medline, CAS, Google Scholar7. Caplen, N.J., J. Fleenor, A. Fire, and R.A. Morgan. 2000. dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene 252:95–105.Crossref, Medline, CAS, Google Scholar8. Boutros, M., A.A. Kiger, S. Armknecht, K. Kerr, M. Hild, B. Koch, S.A. Haas, R. Paro, and N. Perrimon. 2004. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303:832–835.Crossref, Medline, CAS, Google Scholar9. Jaleel, A. and K.S. Nair. 2004. Identification of multiple proteins whose synthetic rates are enhanced by high amino acid levels in rat hepatocytes. Am. J. Physiol. Endocrinol Metab. 286:E950–E957.Crossref, Medline, CAS, Google Scholar10. Sartipy, P. and D.J. Loskutoff. 2003. Expression profiling identifies genes that continue to respond to insulin in adipocytes made insulin-resistant by treatment with tumor necrosis factor-alpha. J. Biol. Chem. 278:52298–52306.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByQuality Considerations in Digital Zero-Price Markets: OECD Background PaperSSRN Electronic Journal, Vol. 900Rheb-TOR signaling promotes protein synthesis, but not glucose or amino acid import, in Drosophila19 March 2007 | BMC Biology, Vol. 5, No. 1Live Cell Approaches for Studying Kinetochore-Microtubule Interactions in DrosophilaRNAi-based tuning of cell cycling in Drosophila S2 cells—effects on recombinant protein yield1 January 2007 | Applied Microbiology and Biotechnology, Vol. 73, No. 5 Vol. 37, No. 6 Follow us on social media for the latest updates Metrics Downloaded 254 times History Received 12 May 2004 Accepted 29 July 2004 Published online 6 June 2018 Published in print December 2004 Information© 2004 Author(s)AcknowledgmentsThis work was funded by National Institutes of Health (NIH) grant no. 1R01GM70851-01.Competing Interests StatementThe authors declare no conflicts of interest.PDF download" @default.
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