RT-PCR

[Andrea]

Hi,

my colleague and me started to do RT-PCR with specific primers for the first time. How can we discern, if our primers are working? We checked already for DNA contamination, RNA integrity and primer integrity and they were all fine.

Cheers,
Andrea.

[thess]

Hi Andrea,

This is the way our lab does it: First, try and run a rtPCR to see whether there are any results at all (use at least two wells per gene and sample to minimize pipetting errors). Before starting the program, add a “dissociation stage”, which will give you peaks indicating the melting point of the PCR product(s). If there is only one peak, i.e. all the produced DNA molecules have the same melting temperature, this is already a good hint that your primers are working fine (and that there is only one cDNA in your sample that fits the primers). Afterwards, run a 3.5% agarose gel with the contents of the same PCR wells for each primer, including a DNA ladder. This shows you the approximate length of the DNA molecules that were produced and, again, if it is only one product.
Hope that helps!
Thess