18S trouble in cleared MMG - (Feb/19/2008 )
I am using eukaryotic 18s from ABI as a housekeeping gene in epithelia-free MMG samples. My gene of interest has been working just fine with the appropriate Ct values (or values we expect). However, the 18s Ct values in many of my samples range from 30-33. We are using the Taqman Gene Expression Analysis kits and master mixes in our lab. My question is, should 18S be this low? When I ran samples from intact MMG (epithelial+adipocyte cells) the 18s was in the teens. Should 18S run approximately the same in different cell types?
P.S. When I did my dilution series my gene of interest curves were between -2.8 and -2.9 ( I used 2 different samples to perform the efficiency curve). The 18s curves did not look good at all because at the different cDNA amounts used there were basically running the same (ie no -3.3 ct difference). I used the same diluted cDNA for both my gene of interest and for the 18s for each sample.
One more thing I would like to add is before I made cDNA I checked my RNA on a gel and found that the 28S and 18S bands looked good. There were no signs of degradation and no genomic DNA contamination.
What type of primers do you use for making cDNA? If it's oligo-dT then 18S is AFAIK not transcribed.
18S usually have a massive expression so it's unlikely it should be low in your sample. Unless you do some treatment that inhibits 18S or have a huge amount of target transript.
What's MMG by the way?