Production of Cyclin D1 specific siRNAs by double strand processing for gene therapy of of esophageal squamous cell carcinoma

Background and aim: (rna interference) is a new strategy in genetherapy and biotechnology which provides new viewpoints in treatment ofdifferent diseases such as cancer and viral diseases. ccnd1 which is a key genein cell cycle is amplified and over expressed in esophageal cancer. the aim of thisstudy was to produce sirnas for ccnd1, the key gene in cell cycle.methods: dsrna digestion method was applied by using recombinant humandicer enzyme to cleave in vitro transcribed dsrna into a pool of 22bp sirna.total rna was extracted and cdna was produced using rt-pcr. t7 promoterwas added to both ends of the dna template by pcr. rna was produced fromboth strands of the dna using t7 rna polymerase. after annealing both strands,dsrna was prepared. finally sirna pool was produced by dicer treatment.results: rna extraction yield from hn5 cell line was 14.69 ?g/106 cell. theresults from beta actin control gene confirmed the cdna integrity. afteroptimization, t7 promoter adding was confirmed using gel electrophoresis anddna sequencing. after optimization dsrna yield was improved. the bestincubation condition was 18h. each microgram of dsrna yielded 0.5 ?g sirna.coclusion: dsrna digestion method includes several steps in which theproduct of each step is used as the precursor for the next step. so optimization andincreasing the specificity and product yield should be the most important goals ofthe study, because the yield of each step has a direct relationship with the finalproduct yield namely; sirna. optimizing and increasing the yield, dsrnadigestion method could be a rapid, available and profitable method for sirnageneration, and providing large amounts of sirna.