A fluorescent reverse transcription-polymerase chain reaction assay to quantify the lipoprotein lipase messenger RNA

Relative quantitative reverse transcription-polymerase chain reaction (rqRT -PCR), which allows an accurate quantification of the amount of mRNA in sam ples potentially differing in the quality of their RNA preparation, was use d to quantify lipoprotein lipase (LPL) mRNA in ovine adipose tissue. A comp arative evaluation of four rqRT-PCR procedures was carried out. The amount of LPL mRNA was assayed relative to either that of gamma -actin (ACT) or cy clophilin (CYC) mRNA, used as endogenous standard. Independent (INACT and I NCYC procedures) or simultaneous (COACT and COCYC procedures) amplification s have been compared. Fluorescently labelled primers yielded PCR products w hich were quantitatively analysed using an automated DNA sequencer. After o ptimizing the PCR cycle number and verifying that the amounts of ACT and CY C mRNA varied only weakly according to the nutritional conditions studied, we have tested the ability of the four procedures to quantify specific vari ations in LPL mRNA. The repeatability of each step and the overall assay re producibility were also examined. The COACT and INCYC procedures were final ly retained to accurately quantify LPL mRNA in AT from nine underfed or ref ed ewes, and gave highly correlated results (r= 0.98, p<0.01). in addition, significant correlations (r=0.83, p<0.01 and r= 0.92, p<0.01 for COACT and INCYC, respectively) were observed with the LPL activity in AT. (C) 2001 A cademic Press.