AN IMPROVED REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION METHOD TOSTUDY 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 tra nscription-polymerase chain reaction (RT-PCR) amplification. This meth od, which is at least a 100-fold more sensitive than traditional RT-PC R, was used to examine elements of apolipoprotein B (apoB) gene expres sion in Caco-2 cells. A region of apoB mRNA spanning the edited sire w as amplified from pre- and postconfluent Caco-2 cells both under diffe rent growth conditions and following alterations in exogenous lipid fl ux to determine changes in posttranscriptional editing. Apolipoprotein A-IV (apoA-IV) mRNA levels were examined in the same samples. The res ults suggest that apoB mRNA editing increases in Caco-2 cells during g rowth but this response is more variable than previously reported. Add itionally, evidence was found for differential editing of the 14 kb an d 7 kb transcripts. By contrast, there was a consistent growth-related increase in apoA-IV mRNA abundance. Neither apoB mRNA editing nor apo A-IV mRNA abundance was modulated in postconfluent cells in response t o different combinations of exogenous lipid. This method should facili tate the study of apolipoprotein gene expression in Caco-2 cells and o ther situations where the target RNA is limited either as a result of low abundance or limiting tissue sample size.