Determination method of sense sequence based on RT/PCR.
When novel sequences are isolated by differential display and other methods, it seems useful to determine which is a sense sequence at an early stage before further experiments. A novel sequence, named MT-001, which shows enhanced expression in the permanent ischemic rat brain, was isolated by differential display. Based on this sequence, a primer set for both direction was designed. Each primer was used to make a cDNA and PCRs performed with each cDNA and both primers. One primer used in the RT step produced a PCR product at the expected position, but another primer in the reverse direction could not. This result indicated that the primer that made the expected PCR product is antisense.
Tohda
Michihisa
M
Department of Pharmacology, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan. tohdam@ms.toyama-mpu.ac.jp
Watanabe
Hiroshi
H
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002545:Brain Ischemia; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D020869:Gene Expression Profiling; D008297:Male; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D012313:RNA