Determination of changes in specific gene expression by reverse transcription PCR using interspecies mRNAs as internal standards.
A method is described for the determination of changes in gene expression by reverse transcription of the target mRNA followed by PCR amplification of the resulting cDNA (RT-PCR), using the lipoprotein lipase gene as the model system. Known proportions of human and rat adipose tissue homogenates are mixed and are processed together throughout the RT-PCR procedure so that the rat tissue serves as an internal standard for the measurement of human adipose tissue lipoprotein lipase (LPL) in all steps including RNA extraction, reverse transcription and PCR amplification. Taking advantage of the highly conserved sequence of the LPL gene across species, selected homologous regions of the human and rat genes are amplified using the same primer pair and resulting in the same lengths of amplified DNA fragments. The two amplified products are then separated from each other by making use of differences in the position of a restriction site in the two amplified DNA fragments. The method is simple, precise and reproducible and avoids construction of tailored nucleic acids for use as internal standards.
Department of Biochemistry, Faculty of Medicine, Laval University, Québec, Canada.
G
G
Lévesque
B
B
Lamarche
M R
MR
Murthy
P
P
Julien
J P
JP
Després
Y
Y
Deshaies
D000818:Animals; D001483:Base Sequence; D005786:Gene Expression Regulation; D006801:Humans; D008071:Lipoprotein Lipase / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats
D012333:RNA, Messenger; D008071:Lipoprotein Lipase