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Quantitative Real-Time PCR


MIQE Guidelines - Where to start when first planning your QPCR experiment (Minimum Information for Publication of Quantitative Real-Time PCR Experiments).

Gene Quantification Home Page - this site has developed into a very useful QPCR reference web site. It summarizes many of the technical aspects of real-time PCR and has numerous software and reference downloads useful for newcomers and experts alike.

QPCR Listserver  - and its archives. This is a good place to look when trying something new.

geNorm is a very useful collection of VBA macros for Microsoft Excel which can help you determine the most stable reference genes for your experiments as well as help with normalization. 

The Ensembl Genome Browser is the home to various genome databases and is a very good reference when searching for splice site locations, etc.

Digital PCR (dPCR)

The Gene Pi Digital PCR Learning Center is an informative website to learn more about dPCR, including a number of statistical tools, such as how to determine the limit of blank (LOB) and the limit of detection (LOD) of your assay, multiple tutorials, and other useful information.

Digital PCR information page from the Gene Quantification.

HomepageYouTube video explaining droplet digital PCR.

Primer Design

Beacon Designer and its online tutorial - Lighthouse has a license for this program available for use in the main lab to help you with primer/probe design.

In addition to Beacon Designer, there are several possibilities for online design of primer/probe sets for QPCR. Examples of these include:

Primer 3 - online primer and probe design site from MIT's Whitehead Institute.

The online assay design service for the Universal ProbeLibrary from Roche can be used for assays with or without probes.

The RealTimeDesign software at Biosearch Technologies.

A database of optimized RT-PCR primers can be found at realtimeprimers.org, located at the University of Pennsylvania.

PrimerBank  - another public databank for PCR primers designed for gene expression quantification.

Company Sites

LightCycler 480 from Roche Applied Science. Also of interest may be the references and application notes for download. There are examples for relative and absolute quantification, as well as for high resolution melting analysis.

QX100 Droplet Digital PCR system from Bio-Rad.

Applied Biosystem's Sequence Detection Systems where there are a number of documents for download with general information on quantitation, as well as an ever-changing list of pre-developed TaqMan assay reagents (PDARs).

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