Luminex Clinical Diagnostic assays designed on xTAG® Technology enable a large number of clinical specimens to be analyzed quickly, cost-effectively, and accurately. When performing these assays, remembering these few technical tips will help you generate optimal results. (And remember to always follow the package insert instructions for use.)
Tip #1: Prepare the RT-PCR Mastermix and add samples in a timely fashion, keeping the enzyme frozen until ready for use.
The best practice is to keep all reagents cold during mastermix preparation, and the stock enzyme should be kept in the freezer or on ice until ready for use. Keeping an enzyme stable is a critical to keep it in an active state. Since proper folding is necessary for an active enzyme, a concentrated effort needs to be made to keep the enzyme from denaturing (i.e. unfolding). A folded protein relies on many interactions to remain in a native state such as hydrogen bonds, electrostatic and polar forces. Keeping an enzyme cold will help increase the stability as heat may disrupt these interactions resulting in activity loss. In addition, it is ideal to reduce stress including unnecessary force. For example, we recommend flicking, rather than vortexing, concentrated stock enzyme to reduce the potential for inactivity.
Tip #2: Bead Hybridization and Detection.
Limit the exposure of Luminex beads and Streptavidin Phycoerythrin reporter molecules to light while setting up and during the incubation period. Prolonged periods of exposure to light will results in photobleaching of the luminex beads, causing the bead classification to fall-outside the bead mapping zone resulting in failed sample acquisition. Phycoerythrin (PE), a phycobiliprotein harvested from red algae, is a water soluble protein that displays very intense fluorescence. Under intense or prolonged exposure to light, the protein will degrade.
Tip #3: Reduce contamination exposure risk.
Proper work-flow and technique will reduce contamination risk. Set-up the lab with physical separation (mechanical barriers) of areas for: mastermix preparation (in the absence of any sample RNA or DNA templates), control and sample addition, and amplification/detection space. Ideally a unidirectional flow from reagent preparation to sample addition followed by amplification and detection. Proper cleaning (chemical barriers) pre-and post- assay set-up with 10% sodium hypochlorite (bleach) solution, followed by removal of the bleach with ethanol (~70 %) solution will help reduce contamination risk.
In addition to following these helpful tips, proper training will also help provide a solid foundation of assay knowledge and technical tips generating optimal results. If you have any questions, please contact your Field Applications Specialist or Luminex’s Technical Support Team at 1-877-785-BEAD(2323) or [email protected].