Acetic acid denaturing for RNA capillary polymer electrophoresis.

A strong denaturant to cleave intramolecular hydrogen bonds in RNA is required for RNA size separation in a small sample volume (<10 nL). We found that carboxylic acids were strong denaturants for RNA and the RNA separation performance was dramatically improved by capillary electrophoresis with a sieving matrix containing acetic acid. We revealed that the denaturing ability of 2.0 M acetic acid was stronger than that of either 2.5 M formaldehyde or 7.0 M urea by estimating DNA melting temperature. Consequently, we suggested "in-capillary denaturing polymer electrophoresis" as the RNA size separation methodology to simultaneously denature and separate RNA in a small sample volume without conventional in vitro sample preparation before electrophoresis. The baseline separation of RNA with a size of 100-10,000 nt was achieved in 25 min by "in-capillary denaturing polymer electrophoresis" with the running buffer containing 2.0 M acetic acid. The resolution and the theoretical plates of RNA separation peaks were larger than those of the RNA separation in a conventional CGE with in vitro sample preparation by 7.0 M urea. In addition, we detected RNA peaks from the nucleic acids extracted from NIH 3T3 cells without DNase enzyme treatment.