Hi Daniel,

for a RT-PCR positive control to be good it does not need to be highly expressed, it just needs to be stably expressed in between your experimental groups. Typical controls would be actin, tubulin, GAPDH or 18s rRNA. Depending on who you talk with, some will tell you that one is better than the rest but in general one can say that depending on the particular experiment any of the controls listed above could be used. For example, if you are studying the effect of a drug that changes cell metabolism probably GAPDH is not the best choice. There is a number of good recent reviews with details about when different housekeeping genes should be used.

Hope this helps,

Ezequiel

Ezequiel, thank you for your answer.  That makes sense.  My follow up question to you would be: how should I go about examining a specific gene as a positive control?  Should I be designing my own specific primers for the gene I want to use (say, actin for example) and then use that in my PCR? Or are there well-established control primers that I can purchase to use?  

Hi Daniel,

there are plenty of papers out there with proven housekeeping gene primers so there is no need to reinvent the wheel unless the species you are working with is not being study and/or the sequences are not conserved. Rather than buying control primers with proprietary sequences I would just synthesize it as it is much cheaper.

This old paper could be useful if you want to quickly get the sequence for several primers:

Nucleic Acids Res. 2010 Jan; 38(Database issue): D792–D799.

Published online 2009 Nov 11. doi: 10.1093/nar/gkp1005

PMCID: PMC2808898

PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification

Athanasia Spandidos,1,2 Xiaowei Wang,1,2 Huajun Wang,1,2 and Brian Seed1,2,*

Hope this helps,

Ezequiel

Thanks again Ezequiel.  I am still new to working with DNA/RNA and am just getting familiar with the many resources online for working with genes.  I used the Harvard/MGH PrimerBase site to find a nice beta-actin primer and ordered the oligo through my university's core facility and should have it soon to test.  Thanks for the interesting article and I appreciate your help!