Author/Authors :
Mottaghi-Dastjerdi، Negar نويسنده Pharmacognosy and Biopharmaceuticals Department, School of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran Mottaghi-Dastjerdi, Negar , Soltany–Rezaee-Rad، Mohammad نويسنده 1Pharmacognosy and Biopharmaceuticals Department, School of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran Soltany–Rezaee-Rad, Mohammad , Ajami، Abolghasem نويسنده Obstetrics and Gynecology Department, Infertility Ward, Emam Khomeini Hospital, Mazandaran University of Medical Science, Sari, Iran , , Rafiei، Alireza نويسنده , , Abediankenari، Saeid نويسنده , , Gharaee، Eshrat نويسنده Pharmacognosy and Biopharmaceuticals Department, School of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran Gharaee, Eshrat , Hashemi-Sooteh، Mohammad bagher نويسنده Molecular and Cell Biology Research Center, Department of Biochemistry and Genetics, Faculty of Medicine, Mazandaran University of Medical Sciences, Hashemi-Sooteh, Mohammad bagher , Rahbarizadeh، Fatemeh نويسنده ,
Abstract :
BACKGROUND AND AIM: (RNA interference) is a new strategy in gene
therapy and biotechnology which provides new viewpoints in 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 aim of this
study was to produce siRNAs for CCND1, the key gene in cell cycle.
METHODS: dsRNA digestion method was applied by using recombinant human
dicer 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 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.
RESULTS: RNA extraction yield from HN5 cell line was 14.69 ?g/106 cell. 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 ?g siRNA.
COCLUSION: dsRNA digestion method includes several steps in 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 the most important goals of
the study, Because the yield of each step has a direct relationship with the final
product yield namely; siRNA. Optimizing and increasing the yield, dsRNA
digestion method could be a rapid, available and profitable method for siRNA
generation, and providing large amounts of siRNA.
