Posts tagged ‘qPCR’

Bio-Rad Sponsors New Version of MIQE qPCR App for Real-Time PCR and Digital PCR

Bio-Rad Laboratories, Inc. announced that it 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 (LaSalle Beauvais, France) 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.

August 14, 2013 at 3:22 pm Leave a comment

Building a custom PCR assay that conforms to MIQE guidelines

If you’ve been following the news lately, you’ve likely heard about Bio-Rad’s new PrimePCR platform which provides scientists with commercial qPCR kits that are guaranteed to conform to the MIQE guidelines. If you haven’t heard about the PrimePCR platform then you can learn more by reading the article MIQE trouble free posted on the Canadian Biotechnologist.

Below we present you with a video tutorial on how you can use the PrimePCR platform to build a custom assay.

November 19, 2012 at 3:29 pm Leave a comment

PrimePCR News Announcement

Bio-Rad Laboratories, Inc., recently announced the availability of its CFX Manager 3.0 software for real-time PCR data acquisition and analysis. By integrating with Bio-Rad’s new PrimePCR™ assays and panels, enhancing its user interface, and adding new data analysis tools, CFX Manager 3.0 now offers researchers a complete, user-friendly solution for gene expression analysis that covers everything from run setup to generating publication-quality figures.

Most real-time PCR system software involves numerous steps to get runs started and requires data export or Web upload for advanced data analysis. CFX Manager 3.0 combines streamlined run setup and sophisticated data analysis in one software package.

Key benefits of CFX Manager 3.0 include:

Quick run start: intuitive navigation, a new startup wizard, and a streamlined interface let users start collecting data with just a few clicks

Integrated PrimePCR workflow: fully integrated workflow for running and analyzing data files generated using PrimePCR assays

Guided gene expression analysis: guided plate setup wizard creates a gene expression analysis–compatible layout to assist users with experimental setup

Extraction of more information: data plotting options that are particularly well-suited for large data sets include volcano plots, clustergrams, heat maps, and scatter plots

CFX Manager 3.0 is compatible with the CFX96™, CFX96 Touch™, CFX Connect™, CFX384™, CFX384 Touch™, and MiniOpticon™ real-time PCR detection systems.

For further details about CFX Manager 3.0, please visit <a href="Bio-Rad Laboratories, Inc., today announced the availability of its CFX Manager 3.0 software for real-time PCR data acquisition and analysis. By integrating with Bio-Rad’s new PrimePCR™ assays and panels, enhancing its user interface, and adding new data analysis tools, CFX Manager 3.0 now offers researchers a complete, user-friendly solution for gene expression analysis that covers everything from run setup to generating publication-quality figures. Most real-time PCR system software involves numerous steps to get runs started and requires data export or Web upload for advanced data analysis. CFX Manager 3.0 combines streamlined run setup and sophisticated data analysis in one software package. Key benefits of CFX Manager 3.0 include: Quick run start: intuitive navigation, a new startup wizard, and a streamlined interface let users start collecting data with just a few clicks Integrated PrimePCR workflow: fully integrated workflow for running and analyzing data files generated using PrimePCR assays Guided gene expression analysis: guided plate setup wizard creates a gene expression analysis–compatible layout to assist users with experimental setup Extraction of more information: data plotting options that are particularly well-suited for large data sets include volcano plots, clustergrams, heat maps, and scatter plots CFX Manager 3.0 is compatible with the CFX96™, CFX96 Touch™, CFX Connect™, CFX384™, CFX384 Touch™, and MiniOpticon™ real-time PCR detection systems.

November 12, 2012 at 11:06 am Leave a comment

A novel approach for studying a large number of related gene targets in a single experiment

Pathway analysis has become an important application in life science research and drug development. Investigating how individual targets communicate and respond within complex molecular networks has helped elucidate how biological processes function at a cellular level. Academic institutions use network analysis to advance our fundamental understanding of molecular and cellular biology while pharmaceutical companies now apply pathway analysis to the development of next generation therapeutics. With the advent of genomics and personalized medicine, pathway-based analysis will become an even more valuable tool in aiding our understanding and ability to navigate signaling and disease networks. As the body of literature regarding these networks continues to rapidly increase, challenges such as assay design and therapeutics development can utilize this knowledge to increase the chance of success in research and development.

Read “Pathway Curation and Real-Time PCR Panel Design Strategy” to learn how Bio-Rad Laboratories’ pathway curation and ranking strategy ensures that the gene assays present on each real-time PCR pathway and collection panel are the most relevant for gene expression profiling based on differential expression studies and the frequency with which gene targets appear in the peer-reviewed literature.

October 29, 2012 at 2:28 pm Leave a comment

A Sensitive Technique for Probing Small Differences in Copy Number Variation

Cytogenic studies over the past 50 years have hinted at the impact that copy number variations (CNVs) can have on phenotypic traits and disease susceptibility. Given the high incidence and clinical impact of CNVs, a precise, rapid and cost-effective method is needed for high-throughput validation of candidate CNV associations and for subsequent routing deployment in diagnostic settings. The predominant method used to validate CNVs in larger population is real-time or quantitative PCR (qPCR), which measures the relative rates of fluorescence increases during the exponential amplification of target and single-copy reference genes. The accuracy and precision of these measurements can be impacted by multiple factors including differences in amplification rates between the target and reference genes, variations in their amplification rates during qPCR, sampling error due to DNA concentration and analysis errors. Weaver et al. rigorously characterized these factors and found that systemic errors can be addressed by increasing the number of replicates to achieve the desired precision. however, the required number of replicates increases rapidly as finer discrimination is desired, with four replicates required to distinguish a twofold difference and up to 18 replicates to distinguish a 1.25-fold difference.

Read Digital PCR – Probing Copy Number Variations Using Bio-Rad’s QX100 Droplet Digital PCR System to learn more on how droplet digital PCR (ddPCR) can be used to determine small fold differences for higher-order CNV states.

October 15, 2012 at 6:38 pm Leave a comment

MIQE Trouble-Free

Real-Time PCR can be tough. It requires careful planning and much optimization. When it works, you feel great. When it fails…fill in the blank. There are times in our research career when we feel like giving up. Nothing we do seems to yield positive results. Then…along comes a kit. Sure, at first we are wary about using a kit. After all, weren’t we put on this planet to troubleshoot and suffer through tortuous experiments? Alas, many of us quickly overcome that guilt and put our trust and faith in the hands of others. But how do we know that commercially available kits are indeed trustworthy? Perhaps they too will yield erroneous results and lead us down the dark path of non-publishable gobbledygook data. So what do we do? We troubleshoot. We troubleshoot the commercially available kit. The kit that we purchased to avoid troubleshooting! Curses! It’s one thing to troubleshoot my own experimental protocol, but to troubleshoot someone else’s? And one that I paid for nonetheless?

Well, fellow scientists, feel the pain no more. At least not in the world of qPCR. Bio-Rad Laboratories has teamed up with leaders in Real-Time PCR to bring you the most robust, commercial qPCR kit on the market, PrimePCR. Bio-Rad’s PrimePCR™ Assays and Panels have been designed to meet MIQE criteria and incorporate the following key requirements:

  1. high assay specificity without the use of a probe
  2. compatibility with standard assay conditions
  3. avoided secondary structures in primer annealing sites
  4. avoided SNPs in target regions
  5. maximized fraction of transcript isoforms being detected
  6. designed intron-spanning assays whenever possible
  7. used latest release of genome builds and annotation databases

Moreover, the kits have undergone wet-lab validation at the hands of PCR professionals so you don’t have to waste precious time validating and troubleshooting an assay that you spent money on acquiring.

To learn more about Bio-Rad’s Prime PCR Assays read PrimePCR™ Assays: Meeting the MIQE guidelines by full wet-lab validation.

October 9, 2012 at 3:20 pm Leave a comment

Finding the rare mutant allele needle in the wild-type haystack

Studies of biomarkers associated with the onset of and progression of cancers have been fraught with poor selectivity and have failed to detect mutant sequences with abundances of less than 1 in 100 wild-type sequences. The QX100 ddPCR platform offers a simple and robust means to increase the precision and sensitivity of TaqMan assays for rare event detections. Not only are direct counts of template molecules within a sample possible (Pinheiro et al. 2012), but partitioning mitigates the effects of target competition, greatly improving the discriminatory capacity of assays that differ by only a single nucleotide. Here, the benefits for clinically relevant assay targets are demonstrated through the BRAF V600E mutation assay, where the LOD improved >100-fold when performed in ddPCR. This platform should prove invaluable to the molecular diagnostics field in general and to the cancer field in particular for the routine assessment of mutation status. Combined with the high accuracy achieved through the use of tens of thousands of partitions, ddPCR represents a unique platform for extremely precise and sensitive measurements of target molecules.

Click here to learn more on how Bio-Rad Laboratories’ QX100™ Droplet Digital™ PCR System can aid in the detection of rare mutant alleles within a background of wild-type sequences.

October 4, 2012 at 4:05 pm Leave a comment

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