Archive for the ‘Bio-Rad Product Highlight’ Category
Yesterday we told you how scientists at the Fred Hutchinson Cancer Research Center were using droplet digital PCR to accurately quantify microRNA biomarkers in an accurate and reproducible fashion.
As a courtesy to those readers who may have missed it previously, we are reposting a video that was produced a couple of years ago with a comprehensive technical introduction to digital PCR. Please let us know if you have any questions regarding this video or droplet digital PCR in general.
“In the field of circulating microRNA diagnostics, Droplet Digital PCR enables us to finally perform biomarker studies in which the measurements are directly comparable across days within a laboratory and even among different laboratories,” said Dr. Muneesh Tewari, associate member in the Human Biology Division at the Fred Hutchinson Cancer Research Center and lead author of the study.
Challenges in miRNA Quantification
miRNAs are small regulatory RNA molecules with diverse cellular functions. The human genome may encode over 1,000 miRNAs, which could target about 60 percent of mammalian genes. Because they are abundant in many cell types, exist in highly stable extracellular forms, and may provide direct information about disease processes, they are being actively studied as blood-based biomarkers for cancer and other diseases.
Quantitative real-time PCR (qPCR) has been used for the analytical measurement of miRNAs in blood samples; however, researchers have found that qPCR measurements of miRNAs in serum or plasma display unacceptably high interday variability, undermining the use of miRNAs as reliable blood-based biomarkers. An approach that yields more dependable results has therefore been sought by researchers in this field.
Advantages of ddPCR for miRNA Detection
Digital PCR has many advantages over qPCR including the ability to provide absolute quantification without a standard curve and robustness to variations in PCR efficiency across different samples and assays. These and other advantages are embodied by Bio-Rad Laboratories’ QX100™ Droplet Digital PCR (ddPCR) system, which was introduced in 2011.
“We chose to use Bio-Rad’s QX100 Droplet Digital PCR system because it was the first system on the market that could make digital PCR practical from a cost and throughput standpoint for routine use in the lab,” said Dr. Tewari.
To assess the imprecision introduced by each workflow step — serial dilution, reverse transcription (RT), and the preparation of PCR technical replicates — Dr. Tewari and his team conducted nested analyses of ddPCR vs. qPCR on cDNA from a dilution series of six different synthetic miRNAs in both water and plasma on three separate days. In comparison to qPCR, the researchers found that ddPCR demonstrated greater precision (48–72% lower coefficients of variation) with respect to PCR-specific variation
Next, the team performed a side-by-side comparison of qPCR to ddPCR for detecting miRNAs in serum. They collected sera samples from 20 patients with advanced prostate cancer and 20 age-matched male controls and measured the abundance of miR-141, which has been shown to be a biomarker for advanced prostate cancer. Samples were analyzed by qPCR and ddPCR with individual dilution series replicates prepared on three different days. The team found that ddPCR improved day-to-day reproducibility sevenfold relative to qPCR. It was also able to demonstrate differences between case vs. control specimens with much higher confidence than qPCR (P=0.0036 vs. P=0.1199).
“Droplet Digital PCR will allow us to accurately follow serum microRNA biomarker concentrations over time during a patient’s treatment course, something that has been nearly impossible to achieve with real-time PCR,” Dr. Tewari said.
Bio-Rad Laboratories is sponsoring a new version of the MIQE qPCR app. Researchers can use the new version to validate their digital PCR (dPCR) experiments according to the recently published digital MIQE (dMIQE) guidelines (Huggett et al. 2013).
Professors Michael Pfaffl (TU Munich, Weihenstephan, Germany) and Afif Nour (King Abdulaziz University, Saudi Arabia) designed the original MIQE qPCR app to help researchers improve their real-time PCR (qPCR) assay protocols by making it easier to adopt a set of best practices described in an earlier publication: The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments (Bustin et al. 2009). Progress bars in the app show the percentage of assay compliance as each item in the MIQE checklist is completed.
“We were pleased to find that the MIQE qPCR app encouraged our customers to follow the new MIQE guidelines,” said Jean-Pierre Dakkak, a laboratory equipment trading company manager.
“Researchers are really eager to learn how this app can make their life easier,” said Dr. Nour. “They report it instills confidence in the validity of every qPCR or dPCR project.”
The new updates include:
- Project-specific checklists — checklists remove unnecessary items; for example, reverse transcription items are irrelevant in DNA research
- Updated references — researchers can stay current with the latest MIQE and dMIQE literature and qPCR symposiums
- Easy export — users can save their projects and share them with colleagues
The MIQE qPCR app runs on the Apple iPhone, iPod touch, and iPad. It has been downloaded more than 6,500 times in 87 countries. To get your copy, visit http://bit.ly/MIQEapp.
A study published today found that Droplet Digital PCR (ddPCR™) can be used as an accurate and precise method for quality control of next-generation sequencing (NGS) libraries. NGS library QC is essential to optimizing sequencing data yield, thereby increasing efficiency and throughput while lowering cost. The research was published in the in the August issue of Biotechniques.
“While real-time PCR has traditionally been used to quantify libraries, we determined that the only truly accurate way to reproducibly quantify our NGS libraries is with ddPCR,” said Dr. Jason Bielas, lead author and Assistant Member in the Public Health Sciences Division at Fred Hutchinson Cancer Research Center in Seattle, Wash.
Quantifying NGS Libraries and Why It Matters
Various commercial NGS technologies require users to load a precise number of viable DNA library molecules onto the instrument to optimize data yield. Performing a sequencing run with either too many or too few library molecules results in compromised data and sometimes no data at all – wasting sample, expensive reagents, user time, and instrument time.
Moreover, fewer bases might be sequenced if library molecules are not the appropriate length to fully utilize the sequencing platform, thus limiting throughput. Given this, quantifying library molecules and determining fragment size range have become crucial steps in library preparation.
NGS instrument manufacturers recommend quantifying libraries using real-time quantitative PCR (qPCR) and determining their size range using gel or capillary electrophoresis. Each of these has its limitations, though, and the steps recommended to address them, can be time-consuming and expensive.
Advantages of ddPCR for Quantifying NGS Libraries
To simultaneously quantify and determine the size distribution of target DNA with a single ddPCR assay, Dr. Bielas and his team exploited a relationship between droplet fluorescence and amplicon size. They confirmed the accuracy and precision of this method by applying it to NGS library preparation.
The ddPCR assay they designed – known as QuantiSize – was developed using the QX100 ddPCR system from Bio-Rad Laboratories. QuantiSize offers the ability to determine the absolute quantity and the detailed size distribution of target DNA in a single ddPCR reaction well, thus avoiding the drawbacks of other independent quantification and size determination methods.
“Now that we have discovered this new correlation, we can also use ddPCR to extract more information on the characteristics of DNA based on the range of fluorescence that can occur within each droplet,” said Bielas.
Having demonstrated the efficacy of this technique, Dr. Bielas is now planning to leverage the relationship between ddPCR fluorescence and amplicon size to explore mutagenic deletion events in both the human nuclear and mitochondrial genomes.