Posts tagged ‘Sean Taylor’
Did you think that sample prep and primer design was straight forward? What impact can the use of different housekeeping genes as controls have on your results? You will be shocked at what you find out.
In this video, Sean Taylor of Bio-Rad Laboratories explains exactly what you must know to perform RT-qPCR according to MIQE standards.
So you’ve heard all the hoopla about MIQE and how important it is to follow the guidelines when conducting your real time qPCR experiments, (if you haven’t, you better check this out!), but where’s the proof that following MIQE actually makes a difference? After all, qPCR was around for several years before anyone came up with this MIQE stuff. Right? Well…maybe not! As it turns out, qPCR experiments that don’t follow MIQE guidelines can be very difficult for others to reproduce and can even lead to incorrect conclusions in gene expression studies.
In a recently released case study involving breast cancer patients, researchers found that the MIQE guidelines played a central role in obtaining the expected conclusions with a positive control target. The article was written to show readers in a simple, stepwise process how to design a good qPCR experiment that covers the major components of the MIQE guidelines. While each step of the experimental design was found to impact the final conclusion (sample collection, RNA quality and purity and the use of appropriate primers), the most striking result was the impact of reference gene selection on the results. At one extreme, normalization by the commonly used GAPDH and 18S reference genes gave either no significant results of statistically significant data that was opposite to the expected outcome, while other more stable reference genes, (HPRT1 and TBP), gave statistically significant data that supported the conclusions from previously published results with this target.
The study concludes that the application of the MIQE guidelines to qPCR experiments result in reliable, quantifiable and reproducible data. With a growing list of journals that are now requiring the submission of supplemental data supporting adherence to the MIQE guidelines,the publication of qPCR data will become more challenging if they are ignored.
So don’t miss out on significant data. Use MIQE!
To read the case study click here.
As a follow up to our video introduction to droplet digital PCR, we are proud to present you with an advanced video presentation on droplet digital PCR technology. Please have a look and let us know what you think!
As promised in our earlier post on Droplet Digital PCR, we are proud to present the following educational training video “An Introduction to Droplet Digital PCR”, given by award winning field application specialist Dr. Sean Taylor.
Be sure to stay tuned for the advanced training video which will be posted in the near future.
Real time PCR for quantitative analysis of gene expression has become an integral part of many (if not most) research labs around the world. When preparing for qPCR analysis, it is important to choose reference genes than do not vary under experimental conditions and that can be used to normalize your data to accurately reflect changes due to the given treatment. (See: Are you using the right reference genes?)
Choosing the right reference genes can be a very difficult task. Most experts agree that when performing qPCR for gene expression, the more reference genes used, the merrier. Unfortunately, “the more the merrier” is also associated with “the more, the more complicated.”
Fortunately, Dr. Sean Talyor, Field Application Specialist at Bio-Rad Canada has found a great program that makes use of many online databases to help you find the ideal reference genes. In the following tutorial, Sean will show you how to use the Refgenes program for choosing your ideal reference genes. The program utilizes a massive microarray database to help determine gene expression under a variety of experimental conditions.
Classifying and understanding genetic variation between populations and individuals is an important aim in the field of genomics. Many common diseases (diabetes, cancer, osteoporosis, etc.) and clinically relevant phenotypic traits are elicited from the complex interaction between a subset of multiple gene products and environmental factors. High resolution melt (HRM) analysis is the quantitative analysis of the melt curve of a DNA fragment following amplification by PCR and can be considered the next-generation application of amplicon melting analysis. It is a low-cost, readily accessible technique that merely requires a real-time PCR detection system with excellent thermal stability and sensitivity and HRM-dedicated software. However, careful sample preparation and planning of experimental and assay design are crucial for robust and reproducible results. The following guidelines assist in the development of such assays.
Click here for more.