Detection of rare mRNA species in a complex RNA population by blot hybridization techniques: a comparative survey

The detection of very rare mRNA species in a complex RNA preparation by current RNA blotting techniques is not straightforward. To be able to determine the size of mRNA molecules representing 10(-6) to 10(-7) of the total mass of an RNA preparation, a quantitative comparison of the level of detection of denatured mRNA species electrophoretically separated on agarose gels, followed by transfer to either nitrocellulose or diazobenzyloxymethyl (DBM) paper and hybridization to specific cDNA probes was carried out. Different transfer procedures were analyzed. Optimal conditions have been found which allowed the detection of RNA bands containing as little as 5 pg of a specific sequence within a few days of autoradiography following hybridization with highly labeled [32P]cDNA probes. Using this procedure it was shown that the low amounts of alpha-fetoprotein (AFP) mRNA sequences present in adult rat liver are mature AFP mRNA molecules.