Identification and relative quantification of adenosine to inosine editing in serotonin 2c receptor mRNA by CE.
A new method has been developed allowing the identification and relative quantification of different forms of mRNA after RNA editing. This method was applied to the serotonin 2c receptor mRNA that potentially exhibits 32 different forms after adenosine to inosine editing at five different sites located in a row of 13 nucleotides. CE was used to characterize fluorescently labeled ssDNA molecules on the basis of their conformational polymorphism. The relative amount of these 32 mRNA forms has been estimated by measuring the fluorescence intensity of each individual DNA strand. Accuracy of quantification was established by diluting one form into another or into a mixture of cDNA, showing linear and precise proportion of each form (0.06 <SD <0.39%). In mouse brain tissue samples, up to 23 different mRNA forms were characterized and quantified, even for forms representing less than 1% of the mixture.
Poyau
Alain
A
Institut Fédératif de Recherche 41, Ecologie, Génétique, Evolution, Développement de Techniques et Analyse Moléculaire de la Biodiversité, Université Claude Bernard-Lyon 1, Villeurbanne, France.
Vincent
Laurent
L
Berthommé
Hervé
H
Paul
Catherine
C
Nicolas
Brigitte
B
Pujol
Jean-François
JF
Madjar
Jean-Jacques
JJ
D000241:Adenosine / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D007288:Inosine / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D044405:Receptor, Serotonin, 5-HT2C / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D044405:Receptor, Serotonin, 5-HT2C; D007288:Inosine; D000241:Adenosine