Advancing qPCR Through Innovation | TS Digest

Scientists around the world employ quantitative real-time PCR (qPCR) to measure the amount of particular nucleic acid sequences within a sample. While this technique is rapid, sensitive, and economical, the instrument that they employ directly affects the reliability and accuracy of their data.

Within qPCR systems, reaction blocks quickly switch between heating and cooling to obtain the precise temperatures required for nucleotide denaturation, primer annealing, and polymerase-mediated extension. However, the speed at which the block changes temperature affects not only the experiment’s duration, but also the block’s thermal uniformity.¹ Furthermore, many reaction blocks lose heat faster on their edges, leading to temperature variation between sample wells and data errors. 

Although stationary light sources are commonly used in qPCR instruments, they generate different light path lengths across wells in the block and consequently produce discrepancies in the data. Scientists can correct the light path differences between wells by normalizing the target-specific fluorescent signal to the signal of a passive dye, such as the ROX fluorescent dye. However, this reduces the number of sequences they can detect simultaneously using multiplexing.

Researchers require improved qPCR instruments that overcome these challenges, such as the CFX Opus Real-Time PCR System. Using an optical shuttle system to center the light-emitting diodes above each well, the instrument eliminates light path length variation and permits scientists to detect up to five targets per reaction well. This feature along with the uniform thermal performance of its innovative reaction block allows researchers to acquire accurate and reproducible measurements from all wells, while maximizing the system’s multiplexing capabilities. 

Read more about this cutting-edge qPCR instrument.

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