Testing your primers
An important step is to make sure that your primers are amplifying what you think they are amplifying. Before running an actual experiment with your samples, you must test your primers with your samples to make sure they are adequate.
1. Test your primers using a standard curve generated from a pooled mixture of your samples. It doesn't matter which samples you choose. Make a serial 1:2 dilution series of your cDNA. Five dilutions is enough. Include a sixth tube that just has water. If you want, you can add an additional sample of RNA as a genomic DNA contamination control.
2. When you perform your run, include a dissociation curve to test for multiple products.
Above is an output from a dissociation curve. A dissociation curve takes your samples after 40 cycles and slowly heats them to find where the melting temperature is. SYBR dye only binds and fluoresces when bound to double-stranded DNA. Therefore, as you raise the temperature, you will reduce fluorescence because you are removing the double-stranded DNA. Each piece of double-stranded DNA has a different melting temperature - the temperature at which double-stranded DNA will separate. If there is only one product in your sample, you most likely will only have one peak. The graph shown above shows a good dissociation curve. A bad one will have multiple peaks.
3. Run a RNA sample to check for genomic DNA contamination. If you have designed your primers to overlap an intron/exon site, they will not work on genomic DNA.
4. Run some product on a gel to make sure your product is the correct size. Some labs do not bother to do this step.
5. Subclone your product into a T vector (such as pGem T-easy kit from Promega), and sequence ten different plasmids to make sure that you product is correct. This is the only way to make sure that the product you are amplifying is really the correct one. However, this is a step most labs do not take. You might be better off designing two sets of primers and verifying similar expression.
TaqMan primers eliminate the need for testing amplifications because they use a third primer. Even if the other two primers can amplify a product, the third primer must be degraded by the 5'->3' exonuclease activity of DNA polymerase to release the two fluorescent probes that result in a signal.