The presence of humic substances and DNA in RNA extracts affects hybridization results

RNA extracts obtained from environmental samples are frequently contaminate d with coextracted humic substances and DNA. It was demonstrated that the r esponse in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. T he decrease in hybridization signal in the presence of humic substances app eared to be due to saturation of the hybridization membrane with humic subs tances, resulting in a lower amount of target rRNA bound to the membrane. T he decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of hi gh amounts of DNA in RNA extracts resulted in membrane saturation. Consiste nt with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced tar get accessibility and membrane saturation effects.