A new PCR-based approach for the preparation of RNA probe.
A number of PCR-based in situ hybridization (ISH) techniques have been reported to facilitate the procedure. However, those techniques require additional gene specific primers with RNA polymerase binding site. We developed a new PCR-based ISH technique without extra gene-specific primers. We amplified gene specific PCR products with regular gene-specific primer pairs. Special linker, including T7 RNA polymerase binding site, was adapted to amplified PCR products. Secondary PCR was performed with T7 primer, and forward or reverse primer, used for the first PCR to prepare template DNA for RNA transcription. We were able to generate sense and anti-sense probes for ISH in a day. Recently, real-time PCR and ISH are required to validate microarray results quantitatively and qualitatively. This technique can be expected to facilitate the high-throughput validation of transcripts detected by microarrays.
Suzuki
Takuya
T
Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
Akimoto
Masayuki
M
Mandai
Michiko
M
Takahashi
Masayo
M
Yoshimura
Nagahisa
N
D000818:Animals; D017403:In Situ Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015347:RNA Probes / Q000138:chemical synthesis / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000166:cytology / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics
D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger; D012243:Rhodopsin