Real-time PCR: more wells please

An Industry Insight feature with Bio-Rad Laboratories

“From an experimental design perspective, a 384-well instrument results in more flexibility and better data”
-Professor Jo Vandesompele

Over the last decade, real-time PCR has secured a position as a mainstay in the molecular biology laboratory for a variety of applications, including gene expression analysis, nucleic acid quantification, pathogen detection and SNP genotyping. In just ten years after the commercial availability of real-time PCR technology, it has continued to evolve in order to keep up with the ever-increasing demands of researchers for easier to obtain, faster results.

Bio-Rad Laboratories released the iCycler iQ TM real-time PCR system in 1999, providing researchers with the first upgrade path from a thermal cycler to a real-time PCR system. At the time, it was the first real-time PCR system capable of generating a thermal gradient across the thermal cycler block and capable of multiplexing up to four fluorophores in a single well. While performing real-time PCR in a 96-well block format has become a recognised standard in the laboratory, the need for higher throughput instruments has increased in recent years. In 2008 Bio-Rad will release the CFX384 TM real-time PCR detection system to accommodate researchers wishing to perform higher throughput real-time PCR without sacrificing the convenience and flexibility available on some existing 96-well real-time PCR systems.

For some, just thinking about running a 384 well real-time PCR experiment raises both technical and psychological barriers. Alternative high throughput techniques such as microarrays have been explored, but these alternatives do not provide the same dynamic range of detection or consistency across experiments. In some cases, laboratories may use multiple 96-well real-time PCR systems to match the throughput of a 384-well system, while still maintaining their comfort level for experimental setup and data analysis.

Today many users are preparing to evolve to the 384-well format. We asked Professor Jo Vandesompele from the University of Ghent in Belgium, and one of the co-founders of BioGazelle, a company that provides software for real-time PCR data analysis, to discuss some of the important considerations that need to be taken into account when performing real-time PCR in a 384-well format.

Bio-Rad: How your lab has adapted to run experiments in a 384-well format?
Prof. JV: We currently have all 3 different 384-well instruments available on the market, including the CFX384 from Bio-Rad, in our laboratory. These systems are mainly used for high-throughput gene expression and gene copy number analysis. Typical experiments include 20 to 400 genes measured in 50 to 500 samples. It is important to realise that 384-well instruments are not only suited for high-throughout experiments; smaller experiments also fit perfectly in a 384-well plate.

Bio-Rad: What were some of the challenges you faced in moving your experiments onto the 384 well system formats and how did you overcome those challenges?
Prof. JV: There was some kind of psychological barrier to pipette into 384-well plates. However, once accustomed to the smaller wells and higher number of wells, people in the lab started to achieve perfect manual pipetting into these plates. We recently introduced a 96-well pipetting system that does most of the liquid handling for the high-throughout experiments.

Bio-Rad: What are some recommendations you would make for working with a 384 well instrument, ranging from experimental setup to data analysis?
Prof. JV: From an experimental design perspective, a 384-well instrument results in more flexibility and better data, as there is, in principle, less inter-run variation. We also like 384-well instruments for the small reaction volumes, resulting in a significant reduction in costs. As the data dimensionality increases, there is definitely a need for automated, flexible and powerful data-analysis. Our qBasePlus software ( http://www.qbaseplus.com ) is perfectly suited to do this job. It really saves us a lot of time, and returns accurate results, moving us in the right direction for future research.

Bio-Rad: Have you seen a trend in Biogazelle software downloads that suggests 384-well real-time PCR is of substantial growing interest?
Prof. JV: 384-well capable instruments make 24% of the market according to our customer base. We have witnessed a steady increase in the use of 384-well capable instruments. We have to acknowledge that there might be a bias in our customer base, as people using a 384-well instrument are typically in need of dedicated data-analysis software.

About Prof. Jo Vandesompele

Jo Vandesompele has been Professor in functional genomics and applied bioinformatics at Ghent University since 2007. One part of his research programme is dedicated to methodological and technical developments in the field of real-time qPCR (bioinformatic tools such as geNorm, qBasePlus ). Jo is the co-founder of BioGazelle, the Real-Time PCR data-analysis company.

CFX384 TM Real-Time PCR Detection System

Designed for the Way You Work
The CFX384 real-time PCR detection system brings flexibility and ease of use to researchers performing high-throughput real-time PCR in a 384-well format. The solid-state optical components provide sensitive detection for precise quantification and reliable performance. The CFX384 system offers a single-fast scan option for data acquisition and four-target multiplex reactions capabilities. CFX manager TM software provides numerous features and tools to streamline data management, from experiment setup to analysis. Enter or edit well information on your own time – before, during, or after a run. CFX Manager software offers enhanced data analysis modules, including gene expression analysis by relative quantity (ΔCt) or normalized expression (ΔΔCt) with multiple reference genes and individual reaction efficiencies used in the calculations. For laboratories operating in regulated environments CFX Manager software, Security Edition offers tools for compliance with U.S. FDA 21 CFR Part 11 regulations.

For more information, please click here.