Hello,

I think it could be a problem with your cDNA preparation, as the CT of B-actin should be at or below 20. You should also run some of the RNA on a gel to make sure that it is properly intact before the reaction, showing different bands for 28S and 18S rRNA. It will also verify the portion of RNA used in the cDNA reaction. Or maybe because of DNA contamination in your RNA sample. It is important because housekeeping gene like B-actin usually has the tendency to highly expressed because of genomic processed pseudogenes which can also amplify from the contaminating genomic DNA present in the sample. Please check, if DNA contamination of your RNA is a problem.

Good luck!

Dear Eric,

I suggest that you verify all the steps that you did including the quality check, excluding each time one possible source of this problem.

Try different quality checks such as the one suggested by Debasish (Running RNA on a gel) or absorbance (260 and 280 nm).

Try using different housekeeping genes to see if the problem persists.

Make sure your primers are specific and they do not amplify something else (Melting curve, sequencing) and that they are sufficiently efficient (~95-105%).

Verify the Quality of your extraction, Reverse transcriptase reaction, Primers, and PCR.

I included in my answer some interesting articles that may come useful to your case.

MIQE Guidelines:

Article The MIQE Guidelines: minimum Information for Publication of ...

Interesting articles related to your question:

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Article Determinants of RNA quality from FFPE samples

Sincerely,

Beside everything mentioned above I would recommend to check B-actin primers first. I've experienced once very similar problems with RNA from cell line and figured out that B-actin (only this particular ones) diluted (10uM ) primers are not very stable compare to other ones after couple freezing/thawing cycles.

Also make sure you use exactly the same MasterMix ( without primers) for all real time PCR reactions. Taq polymerase tends to be oxidased fast and loses its activity upon thawing/freezing, even opening tube, so use of different MasterMixes (thawed/frozen different number of times) may result in different efficiences of real time PCR reaction and hence different Ct values for the same cDNA.

RNA degradation and large amount of dead cells in tissue could be another reason. Check both A260/280 and A 260/230 ratios, RNA integrity as mentioned by others already- this will help to figure out where the problem is coming from.