RNA analysis by MEKC with LIF detection.
We have developed and validated a procedure of high sensitivity for the analysis of RNA. The procedure is based on the separation and detection of the 5'-monophosphates of ribonucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using CE with LIF. BODIPY conjugates of the four common ribonucleoside-5'-monophosphates were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions. After digestion of RNA or oligoribonucleotides to 5'-monophosphates by nuclease P1 and fluorescence labeling BODIPY conjugates were detected and resolved by CE-LIF without further purification steps. Comparative CE-LIF analyses with DNA digested to deoxyribonucleoside-5'-monophosphates showed that the assay is equally efficient and sensitive for RNA analysis. Conditions to determine the modified ribonucleosides inosine, xanthosine, pseudouridine and 2'-O-methyladenosine were also established. The limits of detection were in the range of 80-200 pM. After calibrating the assay with oligoribonucleotides, pseudouridine was quantified in total RNA of Drosophila, human liver, human kidney and t-RNA of Saccharomyces cerevisiae. These studies demonstrate good potential of fluorescence labeling of ribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF to determine RNA composition with high accuracy and sensitivity.
Cornelius
Michael G
MG
Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany.
Schmeiser
Heinz H
HH
D001896:Boron Compounds / Q000737:chemistry; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000032:analysis; D005029:Ethylenediamines / Q000737:chemistry; D007834:Lasers; D012313:RNA / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
C503730:4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine; D001896:Boron Compounds; D005029:Ethylenediamines; D012313:RNA; D004247:DNA