Large-scale, PCR-based gene analysis is only possible when signals are generated, captured, and analyzed accurately and reproducibly. Thermal control, optical readout, and the underlying software algorithms used to measure genetic signals must be precise and reliable from both qualitative and quantitative perspectives. The LightCycler 1536 System from Roche Applied Science addresses these issues using a 1,536-well plate to perform high-speed analyses in an array-like format.
The instrument uses a thermal cycler module for heating and cooling miniaturized qPCR reactions in a 1,536-well plate. Two excitation filters and two detection filters are optimized for detecting green intercalating dyes and monocolor and dual-color hydrolysis probes. This dual-detection format optical read-out is designed to reduce high-throughput layout complexity.
The system software allows integration into automated PCR workflows for flexible data management and allocation in network and LIMS environments. Real-time PCR crossing point (Cp) values, as well as endpoint and slope values, are generated for high-throughput gene expression and genetic variation analyses. Heatmap data output permits quick evaluation of the up to 1,536 results produced in a single run.
The system’s multiwell plates handle low-volume PCR reaction volumes of 0.5 µL to 2 µL. The plate technology uses composite layers of two custom-blended polymers for thermal accuracy, speed, and temperature uniformity. The heat-conducting lower layer effectively thermalcouples the block to the liquid. The upper layer serves as a heat barrier for stable and uniform heating across all wells of the plate. The system uses Thermacore’s Therma-Base technology for fast well-to-well temperature homogeneity.
A high-resolution optical focal length is used to capture and quantify fluorescent real-time PCR signals. High signal excitation and reproducible data acquisition eliminate the need for passive reference dyes―such as ROX―making calibration assays unnecessary.
New RealTime ready reagent chemistry is designed to meet the challenges posed by automated high-throughput PCR workflows. The ready-to-use hot start PCR reagents, which are compatible with fast PCR protocols, make low-volume, automated, high-throughput PCR possible.
The reagents offer high precision for volumes as low as 0.5 µl, with room temperature stability—at least 48 hours—for the longer processing steps required by high-throughput automation. The reagents also enable proprietary error tracking surveillance for quality control verification during automated PCR setup.
DNA master kits are available for both the hydrolysis probe and intercalating dye detection formats. Rapid PCR reaction volume upscaling and downscaling permit straightforward assay adaptation to meet varying throughput needs. For gene expression analysis, DNA probes master reagents are available to eliminate assay optimization.
The system increases single-run throughput fourfold compared to using a single 384-well plate, generating 1,536 results in less than 50 minutes. Low-volume PCR mix miniaturization substantially cuts the cost per reaction by reducing the need for the required enzymes, primers, and fluorescent-labeled probes.