An improved reverse transcription-polymerase chain reaction method to study apolipoprotein gene expression in Caco-2 cells.
We report a method for the detection and analysis of apolipoprotein B mRNA using the thermostable enzyme rTth to perform coupled reverse transcription-polymerase chain reaction (RT-PCR) amplification. This method, which is at least a 100-fold more sensitive than traditional RT-PCR, was used to examine elements of apolipoprotein B (apoB) gene expression in Caco-2 cells. A region of apoB mRNA spanning the edited site was amplified from pre- and postconfluent Caco-2 cells both under different growth conditions and following alterations in exogenous lipid flux to determine changes in posttranscriptional editing. Apolipoprotein A-IV (apoA-IV) mRNA levels were examined in the same samples. The results suggest that apoB mRNA editing increases in Caco-2 cells during growth but this response is more variable than previously reported. Additionally, evidence was found for differential editing of the 14 kb and 7 kb transcripts. By contrast, there was a consistent growth-related increase in apoA-IV mRNA abundance. Neither apoB mRNA editing nor apoA-IV mRNA abundance was modulated in postconfluent cells in response to different combinations of exogenous lipid. This method should facilitate the study of apolipoprotein gene expression in Caco-2 cells and other situations where the target RNA is limited either as a result of low abundance or limiting tissue sample size.
Giannoni
F
F
Department of Medicine, University of Chicago, IL 60637.
Field
F J
FJ
Davidson
N O
NO
D001054:Apolipoproteins A / Q000235:genetics*; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D002277:Carcinoma / Q000235:genetics; D003110:Colonic Neoplasms / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D001054:Apolipoproteins A; D001055:Apolipoproteins B; D012333:RNA, Messenger; C022205:apolipoprotein A-IV