Mouse brain full-length cDNA library construction by negative selection of intact mRNAs.
Synthesis of full-length cDNA libraries is an essential step for the study of gene function. The method for selecting the intact mRNA directly affects the number of full-length transcripts. We have developed a novel method for intact mRNA selection based on the elimination of uncapped mRNAs. A negative-selection strategy that removes both uncapped mRNA and other non-mRNA molecules that present a phosphate at the 5'-end has been applied in the mRNA purification procedures. Briefly, after performing a standard mRNA purification, a biotinylated oligoribonucleotide is ligated to the 5-end phosphate of uncapped mRNAs. Streptavidin extraction is then performed to remove truncated and non-mRNAs from the intact mRNAs. By comparing random sequencing results of mouse brain full-length and standard cDNA libraries, there was a significant increase of full-length clones with the modified procedure. The results showed that the full-length library contained more than 68% full-length clones with the 5'-end positions ranging between -485 to +100 compared to the standard library with 33% of full-length clones and 5'-end positions ranging between -233 to +100. The data were analyzed using the t-test with the significance level set at p<0.05. The statistical results showed that there were significant differences between two libraries in both 5'-end position and mRNA size (p<0.05).
Wu
Ning
N
Center for Biotechnology Research and Education, Langston University, Langston, OK, USA. nwu@luresext.edu
Wu
Huijuan
H
Li
Yandong
Y
Matand
Kanyand
K
D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry* / Q000378:metabolism; D015723:Gene Library; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D012315:RNA Caps; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*
D018076:DNA, Complementary; D012315:RNA Caps; D012333:RNA, Messenger