A modified method for the detection of differentially expressed mRNAs without using radioactivity.
We present here a modification of the original differential display approach using a single oligo(dT) primer for the reverse transcription reaction (instead of the various oligo(dT)NM primers that subdivide the pool of mRNAs) and a combination of 25-mer or 26-mer arbitrary primers together with 30-mer anchored primers for the PCR reaction. The PCR products are, then, efficiently separated in a non-denaturing polyacrylamide gel and the bands are visualized after staining with silver nitrate. The model for the development of our differential display approach was seven clones of an insect species: the aphid Myzus pesicae (Sulzer) (Homoptera: Aphididae). We believe that our modified differential display technique, with the efficient resolution of the DNA bands in a non-denaturing gel and staining with silver can be applied as an alternative non-radioactive detection of differentially expressed messages in various cell populations. In addition, the method could be used as a supplementary tool to other techniques for examining inter- and intraspecific genetic variation in aphids.
Margaritopoulos
J T
JT
Laboratory of Entomology and Agricultural Zoology, Faculty of Crop and Animal Production, University of Thessaly, Fytokou St., 38446, Nea Ionia, Magnesia, Greece.
Kouretas
D
D
Tsitsipis
J A
JA
D000818:Animals; D001042:Aphids; D017931:DNA Primers; D020869:Gene Expression Profiling / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D011851:Radioactivity; D016622:Silver Staining / Q000379:methods*
D017931:DNA Primers; D012333:RNA, Messenger