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Rna interference of annexin II gene in PC3 cells by using small interference RNA synthesized with in vitro
Ya-Wei Yuan1, Ai-Min Sun, Ying Lui
1Department of Radiotherapy, Nanfang Hospital of Nanfang Medical University, Guangzhou 510515. yuanyawei@fangliao.com
Objective:
To silence annexin II gene expression by using small interference RNA (siRNA) in prostate cancer cell line PC3.
Methods:
For in vitro transcription, four sequences of 29-nucleotide DNA template oligonucleotides were designed, and one pair of the sequences were complementary to annexin II gene. The other pair was negative control. The 8 nucleotides at the 3' end of each oligonucleotide were complementary to the T7 Promoter Primer. The sense and antisense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. The siRNA was transfected into prostate cancer cell PC3. For assaying the efficiency of siRNA, confocal microscopy, Northern blotting, and Western blotting were employed to examine the expression of annexin II protein and its mRNA. 3H thymidine was used to measure DNA synthesis.
Results:
The siRNA sequence specific to annexin II gene was capable of inhibiting the expression of annexin II protein and its mRNA. And cellular DNA synthesis was significantly reduced in siRNA transfected cells.
Conclusions:
The protocol for the synthesis of siRNA by T7 RNA polymerase is feasible. Annexin II might be involved in DNA synthesis.
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