Gene expression assay in blood and various tissues using a single-tube real-time reverse transcription-polymerase chain reaction method using an oligodeoxythymidine-immobilized polymerase chain reaction tube.
A single-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) method has been developed which makes it possible to conduct the entire procedure, from nucleic acid extraction to product detection, in a single PCR tube. In this study, we developed the method using an oligodeoxythymidine-immobilized PCR tube, which enables simple and rapid mRNA extraction and quantification of target genes in blood and other tissues. The beta-actin gene was analyzed from lysates of blood and various tissues using this method. The data showed a good correlation between the plotted threshold cycle values and log(10) of blood and tissue amounts without a reduction in PCR efficiency. Gene expression of interleukin-1beta in blood from lipopolysaccharide (LPS)-stimulated rats and of beta(3)-adrenoceptors in adipose tissue from SHRSP.Z-Lepr (fa)/IzmDmcr (obese SHRSP) rats was also analyzed using the single-tube method, as well as a general real-time RT-PCR method, using RNA purified with a silica membrane column. In both methods, the copy number ratio of interleukin-1beta to beta-actin in LPS-stimulated rats was higher than in control rats, and the ratio of beta(3)-adrenoceptors to beta-actin in obese SHRSP rats was lower than in lean littermates. These results indicate that the single-tube method can provide results equivalent to those from general real-time RTPCR methods in gene expression analysis.
Harikai
N
N
School of Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Hyogo, Japan. harikai@mukogawa-u.ac.jp
Saito
S
S
Tanaka
A
A
Kinoshita
K
K
D000199:Actins / Q000235:genetics; D000818:Animals; D053583:Interleukin-1beta / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*
D000199:Actins; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D022702:Receptors, Adrenergic, beta-3