buffer containing detergent. With this approach, approximately 30-40 ng of mRNA can be obtained from 1 ^g of total RNA.
There are limitations to the isolation of polyA RNA using oligo dT or dU. The efficiency of polyA and polyU binding is variable. Secondary structure (intrastrand or interstrand hydrogen bonds) in the target sample may compete with binding to the capture oligomer. Also, mRNAs with short polyA tails may not bind efficiently or at all. AT-rich DNA fragments might also bind to the column and not only compete with the desired mRNA target but also contaminate the final eluant. Potential digestion of the oligo-conjugated matrices precludes the use of DNase on the RNA before it is bound to the column. Treatment of the eluant with RNase-free DNase is possible, but the DNase should be inactivated if the mRNA is to be used in procedures involving DNA components. Furthermore, rRNA can copurify with the polyA RNA. The final purified product, then, is enriched in polyA RNA but is not a pure polyA preparation.
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