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

Abstract

Relative quantitative reverse transcription-polymerase chain reaction (rqRT-PCR), which allows an accurate quantification of the amount of mRNA in samples potentially differing in the quality of their RNA preparation, was used to quantify lipoprotein lipase (LPL) mRNA in ovine adipose tissue. A comparative evaluation of four rqRT-PCR procedures was carried out. The amount of LPL mRNA was assayed relative to either that of γ-actin (ACT) or cyclophilin (CYC) mRNA, used as endogenous standard. Independent (INACT and INCYC procedures) or simultaneous (COACT and COCYC procedures) amplifications have been compared. Fluorescently labelled primers yielded PCR products which were quantitatively analysed using an automated DNA sequencer. After optimizing the PCR cycle number and verifying that the amounts of ACT and CYC mRNA varied only weakly according to the nutritional conditions studied, we have tested the ability of the four procedures to quantify specific variations in LPL mRNA. The repeatability of each step and the overall assay reproducibility were also examined. The COACT and INCYC procedures were finally retained to accurately quantify LPL mRNA in AT from nine underfed or refed 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.