en ايمونولوژي Immunology پژوهشي Research <p><b>Background:</b> RNAi (RNA interference) is a new strategy in gene therapy and biotechnology which provides new promises in the treatment of different diseases such as cancer and viral diseases. CCND1 which is a key gene in cell cycle is amplified and over expressed in esophageal cancer. The objective of this study was production and siRNAs for CCND1, the key gene in cell cycle.<br> <b>Materials and Methods:</b> We used dsRNA digestion method by using recombinant human dicer enzyme to cleave <i>in vitro</i> transcribed dsRNA into a pool of 22bp siRNA. Total RNA was extracted and cDNA was produced using RT-PCR. T7 promoter was added to both ends of the DNA template by PCR. RNA was produced from both strands of the DNA using T7 RNA polymerase. After annealing both strands, dsRNA was prepared. Finally siRNA pool was produced by dicer treatment.<br> <b>Results:</b> RNA extraction yield from HN5 cell line was 14.69 &micro;g/106cell. The results from beta actin control gene confirmed the cDNA integrity. After optimization, T7 promoter adding was confirmed using gel electrophoresis and DNA sequencing. After optimization dsRNA yield was improved. The best incubation condition was 18h. Each microgram of dsRNA yielded 0.5 &micro;g siRNA.<br> <b>Conclusion:</b> dsRNA digestion method includes several steps which the product of each step is used as the precursor for the next step. So optimization and increasing the specificity and product yield should be in the most important goals of the study, because the yield of each step has a direct relationship with the final product yield which is siRNA. Optimizing and increasing the yield, dsRNA digestion method could be a rapid, available and profitable method for siRNA generation, providing large amounts of siRNA.</p> dsRNA digestion, siRNA, RNAi, dsRNA, esophageal cancer, CCND1 10 15 http://rmm.mazums.ac.ir/browse.php?a_code=A-10-25-2&slc_lang=en&sid=1 Negar Mottaghi-Dastjerdi 10031947532846005785 10031947532846005785 No Department of Pharmaceutical Biotechnology and Pharmaceutical Biotechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Mohammad Soltany-Rezaee-Rad mohammad.soltany@gmail.com 10031947532846005786 10031947532846005786 No Department of Pharmaceutical Biotechnology and Pharmaceutical Biotechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Abolghasem Ajami ajami36@gmail.com 10031947532846005787 10031947532846005787 Yes Molecular and Cell Biology Research Center, Departm ent of immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Alireza Rafiei raffiei1710@gmail.com 10031947532846005788 10031947532846005788 No Molecular and Cell Biology Research Center, Departm ent of immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Saeid Abediankenari 10031947532846005789 10031947532846005789 No Molecular and Cell Biology Research Center, Departm ent of immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Eshrat Gharaee 10031947532846005790 10031947532846005790 No Department of Pharmaceutical Biotechnology and Pharmaceutical Biotechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Mohammad bagher Hashemi Sooteh 10031947532846005791 10031947532846005791 No Molecular and Cell Biology Research Center, Departm ent of Biochemistry and Genetics, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Fatemeh Rahbarizadeh 10031947532846005792 10031947532846005792 No Department of Biochemistry and Medical Biotechnolog y, School of Medicine, Tarbiat Modarres University, Tehran, Iran