Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display.
We have developed a method for high-efficiency window separation of cDNA display by increasing the specificity of priming in reverse transcription. In the conventional method, two-base anchored oligo(dT) primers (5'dT16VN3', where N is any base and V is G, A or C) are used to make windows for the display of transcripts. However, reverse transcriptase often extends misprimed oligonucleotides. To avoid mispriming from dT16VN primers, we have developed two new technologies. One is higher temperature priming with reverse transcriptase thermoactivated by the disaccharide trehalose. The other is the use of competitive oligonucleotide blockers that hybridize to the non-selectively primed mRNAs, preventing the mispriming from the VN site. These methods were combined to improve restriction landmark cDNA scanning (RLCS), resulting in the elimination of the redundant signals that appear in different windows. This was achieved by the increased specificity of initiation of reverse trans-cription from the beginning of poly(A) sites. This method paves the way for the precise visualization of transcripts to allow expression profiles in individual tissues and at each developmental stage to be understood.
Mizuno
Y
Y
Laboratory for Genome Exploration Research Project, Genomic Sciences Center (GSC), Institute of Physical and Chemical Research (RIKEN), Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074, Japan.
Carninci
P
P
Okazaki
Y
Y
Tateno
M
M
Kawai
J
J
Amanuma
H
H
Muramatsu
M
M
Hayashizaki
Y
Y
D000818:Animals; D003001:Cloning, Molecular; D017931:DNA Primers; D051379:Mice; D009841:Oligonucleotides / Q000037:antagonists & inhibitors*; D012333:RNA, Messenger / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D014199:Trehalose / Q000737:chemistry*