Posts filed under ‘PCR Video Tutorials’
Scientists and journals have been slow to adopt the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines that were established in 2009 to bolster the reliability of real-time PCR (qPCR) and reverse transcription qPCR (RT-qPCR) data. To help boost adoption, Bio-Rad scientists published a brief and practical guide that concisely summarizes the key steps required to produce high-quality, reproducible data for labs conducting RT-qPCR experiments.
“This paper makes a clear and persuasive case for why it is so important to implement the MIQE guidelines by taking each of the major parameters and highlighting the consequences of not implementing quality control procedures,” said Stephen Bustin, one of the scientists who developed the MIQE guidelines.
In the Journal of Molecular Microbiology and Biotechnology article, Bio-Rad’s Sean Taylor and Eli Mrkusich noted that since 2010, only 5% of papers presenting qPCR data have applied the 2009 guidelines. The adoption rate appears to be increasing — a recent MIQE adoption survey in Nature Methods showed a rate of 11% in 2013 — but almost nine out of ten papers published today do not provide the minimum data necessary to be critically evaluated and could therefore include misleading results and conclusions.
“We believe the reason MIQE is not being widely adopted is primarily that techniques used by labs and even by individual lab members are based on teachings from senior scientists or students who have learned from previous labs,” said Taylor. “This has resulted in wide variability in approaches to designing and performing qPCR experiments between and even within labs that have passed from scientist to scientist without critical examination.”
To encourage adoption of the guidelines, Bio-Rad’s new paper uses concrete examples that demonstrate both good and bad practices for RT-qPCR, from experimental design and sample handling to primer validation and reference gene selection. For example, many researchers do not validate their primers because the sequences were sourced from the literature, obtained from other lab members, or from vendors as off-the-shelf assays that may not have been wet-lab validated. This omission is problematic, because the use of unvalidated primers can lead to gene expression data that at best give good results for the target gene and at worst can lead to “incorrect and even opposite conclusions” and sometimes even yield data for the wrong target. The authors detail precisely how primer validation should be performed to avoid these problems.
Bio-Rad has been at the forefront of promoting good PCR practices since 1999 when the company introduced its first qPCR instrument. In 2010, Taylor published an article in the journal Methods outlining the key steps in RT-qPCR data production that “lead to high-quality, reproducible, and publishable data.” More recently Taylor teamed up with researchers at the INRS-Institut Armand-Frappier in Quebec to demonstrate the consequences of choosing the wrong reference gene in the journal Molecular Biotechnology.
The Journal of Molecular Microbiology and Biotechnology article extends Bio-Rad’s commitment to MIQE guidelines.
“This paper continues the exemplary support that Bio-Rad has given, from the very beginning, to the MIQE initiative,” said Bustin. “Bio-Rad has arguably done more than any other real-time PCR company to support, popularize, and help implement MIQE.”
“For the many researchers in highly competitive fields where turnaround time from experiments to publication is critical, this article is a must-read to ensure that the data from this very sensitive assay give results that reflect the true biology of the tested systems,” said 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.
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.