Harnessing qPCR and RT-qPCR in Your Laboratory

qPCRQuantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) are useful tools in laboratories around the world for absolute or relative quantification of DNA or RNA targets from biological samples. Once you understand the basic principles and have optimized the reaction and cycling parameters, these techniques tend to be rapid, accurate and easy-to-perform—makes me wish that qPCR and RT-qPCR were more commonplace when I worked in the lab and used radioactive Northern blots to quantitate my gene of interest. This is clearly another case of “If only I had known then what I know now”. While I’m no longer working in the lab, it’s not too late for you to benefit from a good introduction to qPCR and RT-qPCR, and on January 15, 2013, that’s exactly what one Promega Research and Development Scientist presented: A webinar entitled “Optimize Your qPCR and RT-qPCR Assays with Careful Planning and Design”.

In this webinar, Nadine Nassif covered the basics of qPCR and RT-qPCR such as the various assay approaches (e.g., dye-based versus label-based methods), assay design and optimization, and the importance of template purity and integrity. She then went into more detail, covering tips for multiplexing and data interpretation. I’m not going to rehash the content of this webinar here because you can view a recorded copy of this webinar. Instead I present here a summary of additional resources so that you can harness the full potential of qPCR and RT-qPCR in your laboratory. This includes additional information to address some of the questions asked during this webinar.

First, anyone performing qPCR or RT-qPCR should be familiar with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, which provide authors, reviewers and editors specifications for the minimum information that must be reported for a qPCR experiment to ensure relevance, accuracy, correct interpretation and reproducibility. These guidelines and more information can be viewed at: www.rdml.org/miqe.php and http://miqe-press.gene-quantification.info.

Also, you should be aware of the multitude of resources available for qPCR and RT-qPCR. These include the Gene Quantification web page, which is filled with links to information about all things qPCR and RT-qPCR and includes lists of relevant publications and a link to a web-based qPCR forum. There are subpages to address many of the questions posed during the webinar about topics such as:
Normalization and selecting suitable reference genes
Relative quantitation, including information about performing calculations
Absolute quantitation, including information about performing calculations
PCR efficiency, including information about how to take into account differences in amplification efficiencies for different templates
Reverse transcription analysis
RNA purity and integrity

Other qPCR and RT-qPCR resources include Stephen Bustin’s qPCR bible A–Z of Quantitative PCR (1), the Promega webinar: “Maximize Your Reverse Transcription-qPCR (RT-qPCR) Assays”, an FAQ for RT-qPCR and a number of sites devoted to predesigned qPCR primers and primer design software, such as Primer Bank, RTPrimerDB, Primer3 and Primer-BLAST.

If you think that your research might benefit from some well executed qPCR or RT-qPCR, be sure to check out Nadine’s webinar and the additional resources listed here. You’ll be glad that you did.

Reference

  1. A–Z of Quantitative PCR (Editor: S.A. Bustin ), International University Line, La Jolla, CA, USA.

About the Webinar Series
www.promega.com/webinars/ provides a schedule of upcoming webinars. In addition, there are links to previous webinars, which allow you to view the recording or download a pdf of the presentation. There is also a pdf of additional material available for each past webinar.

To register for a webinar, use the Registration link at: www.promega.com/webinars/ This allows you to view a live webinar and participate in the live chat.

Need a reminder? You can also sign-up for monthly invitations to webinars at the webinars page (see link above). Note: Live chat is only available for live webinars, not recorded webinars.

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Terri Sundquist

Terri has worked as a Scientific Communications Specialist at Promega Corporation for more than 13 years, and prior to that, spent more than 5 years solving problems and answering questions as a Promega Technical Services Scientist. She graduated with B.S. degrees in Chemistry and Biology at the University of Wisconsin—River Falls, then earned her M.S. in Molecular Biology from the Mayo Graduate School in Rochester Minnesota.

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