TaqMan-based, real-time quantitative polymerase chain reaction method for RNA editing analysis.
Abnormal adenosine to inosine (A-to-I) messenger RNA (mRNA) editing has been linked to several disease states afflicting the central nervous system. Here we report an assay to determine RNA editing frequencies at specific sites that is based on quantitative polymerase chain reaction (qPCR) with TaqMan probes. The assay was tested by measuring the frequency of the A-to-I mRNA editing at the Q/R site of the human kainate receptor subunit GluR5 and was compared with two established methods of assessing RNA editing: sequencing of individual clones and restriction analysis. The qPCR assay displayed high sensitivity and reproducibility, demonstrated exceptional discrimination between edited and unedited transcript variants, and proved to have several advantages over the other editing methods. Due to the fact that TaqMan-based qPCR technology can be easily adapted to different editing targets, the increased capabilities afforded by this new technique should facilitate various RNA editing studies that aim to elucidate the role of this process in normal physiology and in disease.
James J. Peters Veterans Affairs Medical Center, Bronx, NY 10468, USA.
Kevin
K
Wong
Rebecca
R
Lyddon
Stella
S
Dracheva
D000241:Adenosine / Q000032:analysis; D001483:Base Sequence; D006801:Humans; D007288:Inosine / Q000032:analysis; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D017397:Prefrontal Cortex / Q000737:chemistry; D017393:RNA Editing; D012333:RNA, Messenger / Q000032:analysis* / Q000302:isolation & purification; D018092:Kainic Acid Receptors / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000241:Adenosine; D007288:Inosine; D012333:RNA, Messenger; D018092:Kainic Acid Receptors