Nonradioactive Northern blot analysis of plant RNA and the application of different haptens for reprobing.
Northern blot analysis using radioactive probes is still the most common technique to determine the accumulation of transcripts in cells and tissues. The main disadvantages of this technique are the possible health hazard, inconvenience during handling, the high amount of RNA target necessary for detection, and difficulties with stripping and reprobing. In this paper, we propose an easily applicable protocol for Northern blot analysis of plant RNA with enhanced sensitivity using digoxigenin-, fluorescein-, or biotin-labeled in vitro transcripts derived from PCR products. Furthermore, we show the rehybridization of Northern blots with fluorescein as a second hapten, avoiding any stripping procedure.
Peterhaensel
C
C
RWTH Aachen, Institute for Biology I, Germany. c.peterhaensee@dkfz-heidelberg.de
Obermaier
I
I
Rueger
B
B
D001710:Biotin; D015152:Blotting, Northern / Q000379:methods*; D004076:Digoxigenin; D019793:Fluorescein; D017343:Genes, Plant / Q000235:genetics; D006241:Haptens; D001467:Hordeum / Q000235:genetics; D009693:Nucleic Acid Hybridization; D018515:Plant Leaves; D016133:Polymerase Chain Reaction; D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000032:analysis*; D018749:RNA, Plant / Q000032:analysis*; D011849:Radioactive Tracers; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D006241:Haptens; D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger; D018749:RNA, Plant; D011849:Radioactive Tracers; D001710:Biotin; D004076:Digoxigenin; D019793:Fluorescein