how quantitative is my experiment ? - (Jan/31/2008 )

Hello everyone,

The experiment I have been doing is the following : from a tissue, extracting the RNA, then preparing cDNAs using random primers. Using appropriate sets of primers, I have then, during the same PCR and from the same amount of cDNA for each, amplified 4 different - but related, i.e belonging to the same family - genes. I have then run an electrophoresis on an agarose gel. One of the amplifications (all have roughly the same size) clearly gives a band of higher intensity than the others.

Considering that I've used different primers (with different Tm (2ºC span), %GC (5% span), but roughly the same length), can I really say that the difference I see on the gel is likely to correspond to a true difference of mRNA synthesis ? At this point, can one consider that the PCR is as efficient for a set of primer as it is for another ?

many thanks

I would say that it could be considered semi-quantitative. I performed a similar experiment using the same primer but DNA from different cell lines and noticed varying intensities so my advisor suggested that I used GAPDH as a type of loading control. All of my cell lines had the same intensity bands for GAPDH but I still saw the varying intensities for the cell lines so she told me that I could consider it semi-quantitative but that it would have to be confirmed by qPCR in order to be sure there is a difference.

People use real time PCR for this sort of experiments.