Real time PCR

Real time PCR and real time RT-PCR are a powerful way to quantify relative amounts of different genomic sequences (e.g. to quantify the amount of different species present in a biological sample) or relative expression levels of different genes. There is a good web site / resource at the University of Munich for quantifying relative gene expression which includes a number of papers and reviews (pdfs) on this topic. This link is http://www.gene-quantification.info/

Here is a powerpoint presentation entitled: What real time PCR can do for you. It is a bit large, so is given in two pieces. The first part introduces the different real time chemistries which include Sybr Green, molecular beacons and Taqman. Taqman chemistry is the one we've used most, though we've done some work with Sybr Green as well. The second part gives some examples of data and data analysis methods that we've used to get the most quantitative data out that we can.

For RT PCR experiments, one important point is how to normalize the genes you are studying. It is common to use a housekeeping gene (e.g. b-actin) or ribosomal RNA as a normalizer. However, see the articles by Bustin ( 2000, 2002). They discuss the many pitfalls in normalization and essentially resort to using RNA quantification in the end. We have tried to use b-actin for developmental series in fishes and found it to work sometimes and not others.

HCGS has an ABI GeneAmp 5700 purchased with help from the Hubbard Marine Program and NSF. This is a single color machine which means that only one product can be detected in each well. If you are comparing multiple genes (or multiple species) you need to set up a separate PCR reactions for each and then add the corresponding primer/probes.

The basic steps to a real time experiment to look at relative gene expression are