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 RETROscriptTM 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 Insulin stimulates double-stranded RNA uptake in Drosophila S2 cells