I was talking about determing the PCR efficiency, so that would be dilutions of the PCR template (cDNA (or plasmid)).
"700-900ng/ul" No problem.
If you run a cDNA, and a 1/10 dilution, and there is a 3.3 cycle increment, this indicates that the PCR is working efficiently. (Unless the first sample is delayed by contaminant inhibition, and the second is delayed by poor PCR efficiency from those primers or the PCR conditions, and the two delays cancel each other out to giv a normal 3.3 cycle increment. That's why it's a bit better to run a more full dilution series so you see the result after contaminants are diluted out.) I normally run all experimental samples diluted 1/10. What efficiency does your software calculate for the dilutions you did? Did the dilution match the result?
"I did a mini-dilution curve (1:8, 1:4, 1:2, 1, 2x template) with my cDNA and got some dilution-responsive inhibition in my HKG" Are you saying even your positive shows inhibition? Oh, so you know the PCR conditions are not good? If it's from contaminant inhibition, then the efficiency should improve on dilute samples, but maybe you need to be more dilute than 1/8. As I said, I normally run all my samples diluted 1/10. I have seen inhibition if I don't dilute. (Also, it pushes the experimental Cp's back so they will be more likely to be in the standard curve range.)
"Not getting anything real in NRT and NTC wells." If you are getting the same bands as your experimental samples in the NRT, but not the NTC, this could indicate genomic contamination. If you have genomic, then your PCR may be failing for lack of RNA template (DNA would be quantitated by OD instead of the RNA). If you get bands in the NTC negative, and it shows up with a short enough Cp to interfere with your experimental PCR results, this indicates that your primers/PCR conditions are not good (?). There should be no contamination in the NTC, right? This would be unrelated to template.
What are the Cp's of the negatives, the spleen, and the experimentals? You have been running undiluted cDNA? How much RNA concentration in RT? How much RT rxn in PCR?
Is it possisble that the cell just doesn't express the genes. (Or expresses a splice your primer doesn't see?) Did you run a positive primer set for your troublesome experimental samples? A gene that should definitley be expressed in sufficient quantity and is PCRed with known good primers? (Or, is that what you thought you were doing?)
I assume the intron is large enough not to be PCRed, and that isn't one of the bands you saw.
I did polyA selection long ago and haven't checked recent protocols, but you probably need a lot of RNA to purify mRNA. Whole RNA is usually good enough to use.
I gotta go now.