Variability and cost implications of three generations of the Roche LightCycler® 480

Abstract

Real time PCR has become a dominant method for the highly sensitive detection of pathogens in clinical material. Real time PCR can generate a fluorescence signal by using fluorescence labelled probes, allowing us to detect and semi quantify the amount of amplified DNA. Here we test the variability of the detection system and cost implications of three different versions of the LightCycler® 480 (LC480), focusing on the intensity of fluorescence and Cq in monoplex and multiplex rtPCRs.For gastro-intestinal pathogens there was no correlation between the intensity of fluorescence and the Cq value in the different LC480 types. For probes with the dyes FAMTM, HEXTM, Cy5 and Red610 a higher fluorescence intensity was seen in LC480 type II and III compared to LC480 type I. After lowering the probe concentration for the Cy5 dye three-fold (from 0.3μM to 0.1μM) the Cq value remains the same and the intensity of fluorescence decreases. For the LC480 type II and III the difference in fluorescence intensity was much more extreme. The concentration of the different labelled probes can be lowered at least six-fold in LC480 type II and III cyclers while maintaining a fluorescence intensity as high as achieved in the LC480 type I with undiluted probe. In conclusion, the strength of the fluorescence signal of the LightCycler® 480 type III is superior to that of LightCycler® 480 types I and II, allowing the use of lower probe concentrations for all dyes, particularly for the dyes Red610 and Cy5. This results in a two thirds reduction in PCR probe costs. Switching to these newer machines for real-time PCR can reduce dye labelled probe consumption and thus reduce costs significantly.

Integrative omics reveals subtle molecular perturbations following ischemic conditioning in a porcine kidney transplant model

“…The reaction plates were run on a Roche Lightcycler 480, with an initial pre-incubation of 37 °C for 30 s, then denaturing at 95 °C for 3 s, followed by annealing at 60 °C for 30 s. Denaturing and annealing was repeated for 40 runs before conducting melt curve analysis. Using Roche Lightcycler analysis software, the noise band (baseline) was altered to exclude any background noise whilst staying within the lower third of the linear section of the curve to gain initial Ct values [24]. Average Ct values for beta-actin were then subtracted from average Ct values of target genes to get dCt.…”