Genomic DNA shouldn't produce that dramatic an effect: you're essentially suggesting that you have MORE copies of the genome than you do copies of your transcript of interest, which might be plausible if your contamination was high and your GOI was a very, very rare transcript, but even then you'd be looking at Cts in the high 20s/low 30s, not 8 or 9.

You could test this relatively easily by looking for a really abundant transcript like GAPDH or actin: if it IS genomic contamination you should get the same Ct for basically every gene.

I think it's more likely you just have waaay too much cDNA, or the machine is borking out for some reason.

How much RNA are you using to generate cDNA (and which kit, using what kind of priming)? And how much cDNA are you using per well? And how does this compare with your protocol for muscle RNA?

Also, what sort of ballpark Ct values were you getting for muscle RNA?

Finally, do your traces (even though they're going exponential at cycle 8) look ok? As in are they comparable in appearance to those from previous runs (esp in terms of fluorescence units)? A lot of qPCR software has a tendency to autoscale your curves so they look at a cursory glance like reasonably nice exponentials but they're actually saturating super early or not really amplifying at all.

Good points John,

I would make sure that the primers you are using are spanning exon-exon junctions. thereby contaminating gDNA should not be amplified in a standard qPCR. Looks like an artifact to me. Liver tissue RNA extraction can be a little tricky maybe you want to optimize your protocol.

What protocol or kit was used for isolation of the RNA from the tissues? Did you try any dilutions or do any quantification of your RNA samples or cDNA samples, and if so how much total cDNA did you load into each well and what was the final volume? What did your A260/280 ratios look like? What are you using as your reference/housekeeping gene, and what did the Ct numbers look like for it? Did you run a water control on your plate to see if there may be some kind of contamination in one of your qPCR reagents rather than the RNA samples themselves?

I believe you need to take a look at the A260-280 ratio when you are quantifying your RNA samples, How Pure is your RNA and how you dilute your samples when setting up the qPCR plate. Plus it helps a lot in this case to have GAPDH as your control while you run qPCR.

For qPCR YOU need a good reference gene (an endogenous gene) for analysis of your gene expression. A lot of people use GADPH , however, I recommend you to research about it because your data will be highly accurate when you are using a stable reference gene that its expression doesn't change much ( less than 5 variations or even less ) in negative controls and positive controls. You always need to include NRT( no reverse transcriptase sample) and NTC( no template control). NRT is basically your negative control during reverse transcription which has only RNA and water and noreverse transcriptase enzyme. This way you can always monitor what's wrong with the experiment. If you have any expression in NRT you can exclude the data for that sample ( showing genomic contamination). Then if most of your NRT wells showing high expression then you need to treat your samples with DNase I more. A best way to avoid genomic contamination is to design primers for exon-exon junction. This way you wont amplify the genomic DNA. Be aware your amplified fragment should be small around 100 bp. Always run a regular reverse transcriptase PCR and check the contamination on gel before qPCR. It doesn't matter if your gene shows expression at 8 or 9 because you need to compare their expression relative to a reference gene however I recommend you to use less cDNA (about 0.5 ul) or reduce the starting amount of RNA ( you can use 500 ng or even less). If you are using higher cDNA conc. you will see some pick for NET and NTC as well however these picks must have the lowest ct values. For gene anslysis you need to calculate ^^ct ( delta delta ct) for gene expression analysis which you can find a good instruction in Bio-Rad protocol which I recommend you to read. Your ^^ct is the expression of your gene. It is better to include a negative control in your experiment and compare the gene expression relative to this negative control ( this is different from reference gene) instead of zero.

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5279.pdf

I had some experience in isolating total RNA from the liver (calves) and muscle (intestinal smooth muscle; dairy cows). I could isolated sucessfully total RNA and reverse transcribed into cDNA without any problems by using Trizol. The ratio of absorbance 260/280 was quite good: between 1.8-2.1. The only point you have to make sure about is how to collect the upper phase for further manipulation, to avoid contamination with the intermediate phase containing DNA. Good luck!