Application of capillary electrophoresis to the post-polymerase chain reaction analysis of rat mRNA for gastric H+,K(+)-ATPase.
We have developed a competitive quantitative RNA/polymerase chain reaction (PCR) method using capillary electrophoresis in the post-PCR analysis for the quantitation of rat gastric H+,K(+)-ATPase mRNA. Analysis with CE allows quick and direct separation, evaluation, and characterization of DNA fragments similar in size, and it offers a convenient way of automatizing the post-PCR analysis. To estimate the magnitudes of different error contributions affecting the accuracy and reproducibility of the results, an analysis of variance was performed using the data obtained from quantitating mRNA levels in 10 different total RNA extracts from the Corpus region of Sprague-Dawley rats. The result showed that the reproducibility of the method of analysis was sufficient for the determination of H+,K(+)-ATPase mRNA levels among animals even when small fluctuations in RNA synthesis are to be measured. A comparison was also made of the mRNA levels of the beta- and alpha- subunits of H+,K(+)-ATPase, and the results were found to be in the same level.
Stålbom
B M
BM
Preclinical R & D, Astra Hässle, Mölndal, Sweden.
Torvén
A
A
Lundberg
L G
LG
D000818:Animals; D001483:Base Sequence; D004355:Drug Stability; D004586:Electrophoresis / Q000379:methods; D017506:H(+)-K(+)-Exchanging ATPase / Q000032:analysis* / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013270:Stomach / Q000201:enzymology*
D046911:Macromolecular Substances; D012333:RNA, Messenger; D012313:RNA; D017506:H(+)-K(+)-Exchanging ATPase