[Preparation of small interfering RNA expression cassette based on PCR technique].
OBJECTIVE
To modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference.


METHODS
The U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference.


RESULTS
The sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection.


CONCLUSIONS
The siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.
Guo
Qiu-ye
QY
Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, China.
Ma
Wen-li
WL
Zhang
Bao
B
Wu
Qing-hua
QH
Yan
Lü
L
Zheng
Wen-ling
WL
D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics*; D012342:RNA, Small Nuclear; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics
D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D016159:Tumor Suppressor Protein p53; C405486:U6 small nuclear RNA