Using Passive Reference Dyes for Normalization and Troubleshooting in qPCR

31 May 2015

Three statements are often made about what the use of a passive reference achieves with respect to normalizing non-PCR related fluorescence variation: It normalizes for pipetting errors; it normalizes for well-to-well optical variations on the instrument; it normalizes for changes in concentration or volume of reaction mix during the qPCR run. This technical note discusses the accuracy of these statements and explores other benefits of using passive reference dyes in qPCR.

PCRmax Eco 48 qPCR system

PCRmax

The PCRmax Eco 48 utilises patented hollow liquid filled silver and gold block and optical technologies to deliver extreme speed, sensitivity and data quality, with optimised 40 cycle protocols completing in as little as 15 minutes, all using standard plastic ware and consumables. This matched up with sensitivity down to 1 copy detection and the world most thermally accurate block the Eco 48 will deliver Speed. Confidence. Performance. Value and Sensitivity. The PCRmax Eco 48 real time PCR system is a high specification, feature rich thermal cycler that accommodates a unique 48-well polypropylene PCR plate utilising the same geometry as standard 384-well plates, but only 1/8 of the size. This enables users to dramatically reduce the qPCR reagent volumes compared to traditional 96-well instruments, whilst still producing a strong fluorescence signal. Due to this small volume the user can also use less precious template per reaction allowing for more assays to be performed. Minimizing the plate size also significantly improves thermal uniformity and as such consistency from run to run and system to system allowing users to pool individual plates together into one large experiment massively increasing the scale of the system and ease of sharing data with colleagues. Eco 48 ready to perform four colour multiplexing, High Resolution Melt analysis and has free or licence MIQE compliant software.

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PCR and Thermal CyclingPolymerase chain reaction (PCR) kits and thermal cyclers are used for the in vitro amplification of DNA permitting subsequent analysis and experimental procedures. Explore a range of high-quality polymerase, primers and nucleotides or simplify your workflow with a PCR mastermix. Find reverse transcription PCR (RT-PCR) and cDNA synthesis kits for RNA products and libraries. Quantitatively measure the amplification of DNA with real-time PCR (qPCR) and droplet digital PCR (ddPCR) kits and systems, and discover automated PCR setup solutions to increase throughput. Alternative DNA amplification methods also include recombinase polymerase amplification (RPA) kits. Find the best PCR kits and thermal cyclers and purification equipment in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.qPCRQuantitative PCR (qPCR) is a molecular biology technique used to amplify and measure the amount of a specific DNA or RNA target. It plays a crucial role in gene expression analysis, pathogen detection, and quantifying DNA or RNA in various applications. Real-time PCR provides precise, quantitative results and is widely used in research, diagnostics, and clinical studies. Browse our peer-reviewed product directory to find the best qPCR solutions; compare products, check reviews, and get pricing directly from manufacturers.
Using Passive Reference Dyes for Normalization and Troubleshooting in qPCR