Different Ct values for housekeeping genes

[Masood]

Hi colleagues

I have recently started running some quantitative PCR for two genes Dicer, Drosha and 2 housekeeping genes B-Actin and GAPDH.

The Ct values of Housekeeping genes were ranging from Ct=25 to Ct=30 for both control and diseased groups. Please note that I used same input RNA for RT step measured 3 times with Nanodrop.

In addition: In some samples the Ct value of Dicer and Drosha are less than the Ct of Housekeeping gene for Example Drosha Ct=22 and B-Actin Ct = 25. In this case can I use
2-ΔΔCt


In advance, thank you for your time and consideration and I look forward to hearing from you.
Masood

[mchlbrmn]

(Disclaimer: I'm not a PCR expert, but...)

Did the two housekeeping genes generally agree with each other? That's the important thing. There are papers on how to evaluate this. I'm not expert on that, so I won't give much advice, but in some cases housekeeping gene selection is important because a good control gene for one experiment, may have variable expression in another case (with different tissues, or cell activations, and etc.)

Also, the reason for using housekeeping genes is that using a constant RNA input concentration in the RNA cannot always control the experiment enough. For example, if genomic DNA is present, the DNA will be quantified along with the RNA regardless of how many times it is repeated. Other contaminants can also be present.

I use a different formula, but I think that if you think it through, or test it on samples, you'll find that it doesn't matter if the experimental Ct is larger or smaller than the housekeeping.

[Masood]

Hi mchlbrmn

Kindly note that you have forgot to answer the below listed point:

In some samples the Ct value of Dicer and Drosha are less than the Ct of Housekeeping gene for Example Drosha Ct=22 and B-Actin Ct = 25. In this case can I use
2-ΔΔCt

best regards
Masood

[relaxin]

Yes, you can.

In your case, ΔCt for Drosha = 22-25 = -3. Depending on the ΔCt values for treatment and control, your ΔΔCt value may be positive or negative. If it is positive, then 2^-ΔΔCt can be determined as 1/2^ΔΔCt.