PCR comparison of gene expression - (Dec/13/2004 )
can someone shed some light on this topic for me... if someone were to set up a PCR based experiment to compare the expression of two different genes.... this would be a real time PCR? and it would require primers specific for those two genes, and another set of primers for a control (ie GAPDH etc)?
is there another way to compare the expression of two genes?
You are right, vetticus.
For expression analysis, any technique that involves amplification is not reliable. Hybridization analysis such as Northern and Western are much better than PCR based methods.
cool, THANKYOU!! green,
what's used in a clinical setting? do pathologists use RT-PCR or nothern analysis? if you've been diagnosed with cancer, and it's been cut out, and a sample is sent of to pathology for it's characterisation... and a part of it is to do an analysis of gene expression..... would they do the quicker, though questionable RT-PCR.... or a time consuming northern? or are they moving on towards microarrays?
That would be a different story if you are talking about clinical setting. Immunohistochemistry or tissue microarray (if there are many samples) would be the way to go. I don't think cDNA microarray at current stage will yield any meaningful data for clinicians to make decisions.
Real time PCR is a validated method for expression analysis of genes, though there are a number of caveats, such as the choice of a control gene, usually people use a gene that has consistent expression throughout the body or tissue of interest such as GAPDH or Beta-actin, though some advocate the use of 18s RNA.
In fact real time PCR has now largely replaced northern or western blotting for expression analysis due to the greater sensitivity of the PCR based system, and papers these days often expect to see real time results rather than the other methods.
Check out this web site for more info: http://dorakmt.tripod.com/genetics/realtime.html