Verification of differential gene transcription using virtual northern blotting.
We introduce here an alternative to conventional northern blotting that requires only minute amounts of RNA. This has been achieved by modification of methods currently used for the mapping of mRNA 5'-terminal ends. The terminal desoxynucleotidyl transferase-mediated G-tailing, cap finder, ligation-anchored and RNA ligase-mediated approaches followed by polymerase chain reaction protocols all produced high quality cDNAs in large amounts. These cDNAs could be separated by electrophoresis to obtain virtual northern blots that could replace conventional northern blots. All the essential information, including transcript length and the expression pattern, are preserved in these cDNAs, even if the transcripts are long or GC-rich. In addition, minute amounts of material (less than 100 cells) are sufficient to produce more than 100 virtual northern blots, making this approach extremely versatile.
D000818:Animals; D015152:Blotting, Northern / Q000379:methods*; D018076:DNA, Complementary / Q000032:analysis*; D006110:Grasshoppers / Q000235:genetics / Q000378:metabolism; D007893:Leishmania donovani / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*