Highly sensitive northern hybridization of rare mRNA using a positively charged nylon membrane.
The effect of different RNA fixation methods using Hybond-N and Hybond-N+ nylon membranes in Northern blot experiments was analyzed with human glyceraldehyde-3-phosphate dehydrogenase and human interleukin-3 receptor alpha-subunit as radioactive labeled DNA probes. Highest sensitivity could be achieved by mild alkaline fixation of RNA to positively charged Hybond-N+ nylon membrane. Fixation by UV light resulted in reduced sensitivity in comparison with alkaline treatment. Fixation of RNA to Hybond-N or -N+ membranes by baking for 2 h at 80 degrees C resulted in a strong decrease in sensitivity and should be avoided when using modified nylon membranes in Northern hybridizations.
Beckers
T
T
ASTA Medica AG Experimental Cancer Research, Frankfurt am Main, FRG.
Schmidt
P
P
Hilgard
P
P
D015152:Blotting, Northern / Q000379:methods*; D015342:DNA Probes; D004563:Electrochemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D009757:Nylons; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis*; D016185:Receptors, Interleukin-3 / Q000235:genetics; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D008567:Membranes, Artificial; D009757:Nylons; D012333:RNA, Messenger; D016185:Receptors, Interleukin-3; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases