Evaluation of RNA messengers involved in lipid trafficking of human intestinal cells by reverse-transcription polymerase chain reaction with competimer technology and microchip electrophoresis

Abstract

We developed a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) procedure based on the combination of competimer technology with microchip electrophoresis. The approach was applied to total RNA extracts from the human colon carcinoma cell line CaCo-2 cultured for 22 days onto tissue plate inserts that allow the polarized cell growth. At time of experiment these cells were incubated for 2 h with lipid micelles containing either cholesterol or a mixture cholesterol/beta-sitosterol or serum-free Dulbecco's modified Eagle medium (DMEM) alone. Total RNA was extracted from cell pellets by silica membrane spin columns and reverse-transcribed by a genetically engineered M-MuLV enzyme and an oligo (dT)(15) primer. The cDNA underwent PCR amplification for the following genes: apolipoprotein A1 (ApoAI), apolipoprotein E (ApoE), ATP-binding cassette A1 (ABCA1), liver X receptor alpha (LXRalpha) and farnesoid X receptor (FXR). The beta-actin gene, used as an endogenous reference, was coamplified with mixtures of primer and competimer at a ratio leading to a reference band of intensity comparable to that of the gene under analysis. Amplification products were separated and quantitated by microchip electrophoresis in order to determine, for each gene, the optimum cycle numbers and primer to competimer ratios for the beta-actin therefore used for evaluating the relative variations of gene expressions in the different experiments. The incubation with cholesterol micelles stimulated both LXR and FXR expression that was accompanied by an increased expression of ABCA1 and ApoAI genes (1.4- and 1.5-fold, respectively) and halved the APOE expression. The effect on ABCA1 and ApoAI expression was even stronger (5.7- and 2.6-fold, respectively) with beta-sitosterol-containing micelles. Similar changes are in line with previous findings and suggest that the short incubation with beta-sitosterol-enriched micelles stimulates a specific mechanism that, via ABCA1 activation, can increase the cholesterol and phytosterol basolateral efflux.