Highly sensitive northern hybridization of rare mRNA using a positively charged nylon membrane

Abstract

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.