SPECIFICITY OF OLIGO (DT)-CELLULOSE CHROMATOGRAPHY IN ISOLATION OF POLYADENYLATED RNA

Nonspecific types of binding (occur when oligo(dT)-cellulose is used to analyze or prepare poly(A)RNA [from mouse brain]. Nonpolyadenylated nucleic acids bind and are eluted under conditions used to elute poly(A)RNA. Tight nonspecific binding occurs in which poly(A)RNA fails to elute under conditions which dissociate A-T bonds. Hydrolysis is required to remove tightly bound RNA. Oligo(dT)-cellulose has a low capacity for both these types of binding, and can be readily preempted with a heterologous RNA, e.g., bacterial. Indirect nonspecific binding can also occur. rRNA aggregates with poly(A)RNA and thus can bind indirectly to ligo(dT)-cellulose. After these aggregates are disrupted by treatment with DMSO [dimethyl sulfoxide] and heat, poly(A)mRNA free of rRNA can be isolated. Efficient recovery of poly(A)hnRNA from total nuclear RNA is accomplished using oligo(dT)-cellulose if the RNA is 1st subjected to conditions which disrupt aggregates and reduce secondary structure. Of the purified poly(A)hnRNA and poly(A)mRNA 95-98% rebinds to oligo(dT)-cellulose.