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Posts Tagged ‘qPCR’
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.
Well…you heard it here first folks. Last week we told you about Bio-Rad Laboratories new line of pre-validated, MIQE friendly qPCR assays and how this is bound to save researchers troubleshooting time and frustration (see MIQE Trouble-Free). Now the story has made the news. According to the story on GenomeWeb, Bio-Rad’s launch of PrimePCR has given the company the distinct advantage of being the only vendor whose entire portfolio of qPCR assays has been validated to such standards. The company expects the assays, which cover approximately 95 percent of the human transcriptome, to save researchers time and resources by obviating the need to validate assays in their own laboratories.
Click here to read the rest of the story (subscription required).
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 (we shouldn’t have to tell you what that is, just see our previous posts “A practical approach to MIQE for the bench scientist” and “Applications of MIQE to real time quantitative PCR” among other posts) and incorporate the following key requirements:
- high assay specificity without the use of a probe
- compatibility with standard assay conditions
- avoided secondary structures in primer annealing sites
- avoided SNPs in target regions
- maximized fraction of transcript isoforms being detected
- designed intron-spanning assays whenever possible
- 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.
“People are different from each other in ways that are fascinating and medically important, and to understand the ways in which variations in our genome give rise to those differences presents an important and interesting set of questions,” says Dr Steve McCarroll, faculty member and principal investigator with the Department of Genetics at Harvard Medical School and the Broad Institute of Harvard and MIT. The McCarroll laboratory studies the biological effects of human genome polymorphism, seeking to define how genome variation influences gene expression and risk of disease. Their work requires them to accurately measure the copy number of a genomic sequence with integer precision in hundreds of people. While real-time PCR and CGH (comparative genomic hybridization) arrays are useful for measuring simple deletions and duplications, these techniques simply weren’t sufficient for obtaining single-molecule resolution of target sequences with extreme precision and accuracy. That’s where Droplet Digital PCR entered the story.
Learn how Dr. McCaroll’s lab, along with other labs across the globe utilize ddPCR in applications such as:
- Absolute quantification of HIV proviral DNA and human genomic DNA in the same reaction
- Absolute quantification of HIV viral RNA using two-step reverse transcription-ddPCR
- Rare event detection of BRAF V600E, EGFR L858R and T790M, JAK-2, and cKIT (cancer mutation analysis and drug resistance mutation)
- Absolute quantification of miRNA from circulating nucleic acids for biomarker identification
- High-resolution copy number variation (CNV) analysis of human and mouse DNA’s
- High-resolution CNV analysis of the MET gene, association study for lung cancer
- Absolute quantification of next generation sequencing library
- Rare event detection, mitochondrial mutagenesis
- Absolute quantification of the BCR-ABL (Philadelphia chromosome) fusion gene and transcript, association with leukemia
- Absolute quantification and sex determination of fetal DNA in circulating nucleic acid purified from maternal blood
For more information read “Third Generation PCR: A Novel Way to Accurately Measure Copy Number Variation in the Human Genome.”