Re-suspend the pellet in 100 ml of nuclease-free water and check the RNA concentration as before but using the following algorithm: A260 x dilution factor x 40 = RNA conc. communication), however appears to have lost this pathway. In tissue culture cell Pinocembrin lines, RNAi has been used successfully in the study of mitosis and cytokinesis (14-20) and the method has become a prominent tool in cell Pinocembrin cycle research. The advantages of using cell lines for RNAi studies of mitosis include the sequenced and well annotated genome with a relatively high genetic conservation with humans (21), the favourable cytogenetics for the study of chromosomal processes (tissue culture cells in the upcoming years (22). However, interpretation of Sh3pxd2a results after RNAi in show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by clusters of centrosomes (23). The use of RNAi in transcription using the previous PCR fragment as template. The lower band corresponds to ssRNA that did not form duplexes. (C) Monitoring of protein levels upon addition of 30 g of dsRNA to the cells by immunoblot using MAST polyclonal antibodies and anti–tubulin monoclonal antibodies as a loading control. (D) Determination of the amount of dsRNA necessary to deplete MAST from S2 and Dmel2 cells. Protein levels were monitored 120 h after addition of dsRNA. It is obvious that for the case of MAST, protein depletion is usually significantly more effective in S2 cells. (E) Addition of dsRNA to S2 cells did not cause a significant effect upon cell viability throughout the experiment. S2 cells can be produced in plastic flasks at 25C in Schneiders Drosophila medium (Gibco) supplemented with 10% FBS. For the RNAi experiment, the cells must be in exponential growth. This can be achieved by diluting the cells 1/5 into new media every 3-4 days. Preparation of dsRNA Drosophila cells respond to the presence of large dsRNAs by shutting down protein translation, but mammalian cells do not. This greatly simplifies the process and lowers the cost of RNAi in Drosophila cells, as it is usually not necessary to purchase custom oligonucleotides or construct complex vectors. Instead, the first step towards to specifically knocking down the expression of a Drosophila protein of interest by RNAi is usually to synthesize a dsRNA fragment of ~700 bp (although we have also succeeded using smaller or larger fragments). For this purpose, we designed ~18mer sequence specific oligos to amplify a PCR product of ~700 bp from your cDNA of interest that is cloned into a plasmid. In order to generate the dsRNA from your PCR products, these primers must incorporate a 5 T7 RNA polymerase minimum binding site (Table ?(Table1).1). Table 1 Primer sequences utilized for the preparation of the PCR product utilized for in vitro RNA transcription of MAST/Orbit. The sequence corresponding to the T7 RNA polymerase-binding site is usually underlined. Primer Sequence Forward TAATACGACTCACTATAGGG GAAGGACGAATAGACATTReverse Pinocembrin TAATACGACTCACTATAGGG TCCTGTTTGACCTGGTCG Open in a separate window The successful choice of the optimal sequence fragment seems to be random, however we have found that starting in the 5-UTR Pinocembrin covering the codon for the first methionine may in some cases improve the specific silencing of the protein of interest. We then prepare 10-12 PCR reactions, each made up of 0.5-1 ng of template plasmid DNA containing the cDNA of interest, 1mM of each primer, 2.5 mM of dNTP mix, Pinocembrin 2.5 U of Taq polymerase (Boehringer), enzyme buffer supplemented with Mg2+ according to the manufacturer and water for a final volume of 100 ml/PCR reaction. We use the following PCR program: 94oC for 2 min (warm start), add Taq polymerase and then do 30 cycles of 94oC C 30 s; 55oC C 60 s; 72oC C 60 s, followed by 72oC for 10 min. The PCR products are purified using the PCR Clean-Up kit (MoBio) according to the manufacturers instructions (3 PCR reactions can be run through each column). We test the efficiency of the PCR by electrophoresis by running 1.