Hi Behnaz Farhadi, when you say it does not change with higher concentrations of cDNA, you mean the Ct-values stay the same or that beta-Actin has still higher Ct-values than your gene of interest? If the Ct-values stay the same with different cDNA concentrations then I think there is something going wrong. Otherwise, a reference gene showing higher Ct-values than your gene of interest is possible and no issue by itself. Did you test other reference/housekeeping genes?

I think the main problem here is that you're getting a Cq of ~28-30 for a gene as robustly expressed as beta actin. As a rule of thumb, a template molecule present at only ONE copy per well usually produces a Cq of ~35. Accordingly, cq values of 30 correspond to maybe 30-50 transcripts, which is really low.

In my hands beta actin usually pops up around Cq of ~20, i.e. about 200-1000 times more abundant than the values suggested by your data.

And you know, if you're using it as a reference, you want it to be pretty abundant, since the more abundant it is, the less subject to stochastic noise it will be.

So my take-home from what you're showing here is "your PCR is not working well", and so you probably need to do some troubleshooting.

How much RNA are you using to make cDNA, and how are you checking it for quality/quantity? What are the 260/230 ratios like?

How much cDNA are you using per well? You should dilute your cDNA (ALWAYS dilute your cDNA), because you hardly need any, and ironically too much cDNA can inhibit your PCR.

How efficient is your PCR? You can test this with a serial dilution of cDNA (if you've established you have a decent sample that gives robust and low Cq values) or just use a purified (gel extracted, cleaned) PCR amplicon at known dilutions. You should have a gradient of ~-3.32, corresponding to an efficiency of 2 (i.e. 100%)

Thanks for your answer John Hildyard

I think cDNA can't be the problem here because my target genes work really well in every qPCR test. I use 6 microliters for cDNA synthesis and I've checked RNA with nanodrop (the concentration was about 1500 ng/ul and the 260/280 was about 2.06) and electrophoresis gel. They were okay without any contaminations.

I diluted cDNA to 1/10 concentration and used 2 microliters per well for qPCR.

I didn't check the efficiency of my PCR.

Is dimer the reason for this result?

I appreciate your help Simon Stuecheli

Yes, I have checked other reference genes such as tubulin, 18S, and two different beta actins and none of them were good. These genes had some reasonable problems while in this new actin that I'm using hasn't been found the problem.

260/230! Not 260/280.

260/280 is for protein contamination (the 'shoulder' on the RNA absorbance curve), but protein contamination is almost always minimal (and often not problematic even if substantial).

260/230 is for guanidium salt contamination, which is a common carryover with most RNA extraction methods. Guanidium salts are chaotropic, and will really, really interfere with cDNA synthesis and PCR.

If, as you say, the rest of your genes work fine and give better Cqs (over the 16-25ish range) then your cDNA is probably fine, but still: worth checking.

Also, 6ul of 1500ng/ul is 9ug of RNA: that's a lot of RNA (most kits cap out at 2ug per reaction, and I typically try to aim slightly below that for safety ~1.6ug). Using too much RNA will saturate your system and probably result in poor cDNA synthesis.

Other than that, the other possible explanation is just "bad primers". Sometimes a given set of primers just works poorly, and the easiest solution to that is to throw them away and design new primers. Primers are pretty cheap, so I usually design two or three pairs to any given GOI, and then just keep the one pair that works really reliably.

Dear John Hildyard

I used the Terizol reagent method for DNA extraction so I have contamination for the 260/230.

I think designing new primers can solve the problem.

I appreciate your time.

For future reference, 260/230 ratios above ~1.7 usually work ok.

Below 1.0 they really won't work. Between 1 and 1.7 you're taking your chances.

You can improve 260/230 ratios substantially by simply reprecipitating your RNA -I've posted a method for this on RG probably ten or so times, so it should be hard to find my protocol (sorry: I realise this is lazy).

John Hildyard

I appreciate your help so much.

It is not uncommon for the Ct value of the reference gene to be higher than that of the target gene. In fact, it can vary depending on the experimental conditions and the nature of the genes being studied. The Ct value of the reference gene should be stable across different samples and treatments. A higher Ct value does not necessarily mean that the gene is less expressed, but it may indicate that the gene has a lower abundance or a different expression pattern compared to the target gene. It is important to use a validated reference gene and to perform appropriate normalization methods to account for differences in expression levels and technical variations. Additionally, it is recommended to perform a melting curve analysis to confirm the specificity of the PCR products.