PCR-based generation of shRNA libraries from cDNAs.
BACKGROUND
The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries; one is to use chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries.


RESULTS
We report here an improved method for constructing genome-wide shRNA libraries enzymatically. The method includes steps of cDNA fragmentation and endonuclease MmeI digestion to generate 19-bp fragments, capping the 19-bp cDNA fragments with a hairpin oligonucleotide, and amplification of the hairpin structures by PCR. The PCR step converts hairpins into double-stranded DNAs that contain head-to-head cDNA fragments that can be cloned into a vector downstream of a Pol III promoter.


CONCLUSIONS
This method can readily be used to generate shRNA libraries from a small amount of mRNA and thus can be used to create cell- or tissue-specific libraries.
Du
Cheng
C
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-0766, USA. cdu@unmc.edu
Ge
Baosheng
B
Liu
Zhongfeng
Z
Fu
Kai
K
Chan
Wing C
WC
McKeithan
Timothy W
TW
D003001:Cloning, Molecular / Q000379:methods*; D053938:DNA Fragmentation / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015723:Gene Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*
D034741:RNA, Small Interfering; D004262:DNA Restriction Enzymes