How much volume RNA are you actually putting into your RT reaction? I suggest doing your dilutions after you do your RT reaction. Dilution of your RNA affects the kinetics of your RT reactions as well as your PCR reactions unless you compensate your primers and enzymes relative to your RNA dilutions. This is more true with hexamer primers than it is with DT oligos or GSPs.

A constant concentration of hexamers across a series of dilution of RNA will produce smaller cDNA products the larger your dilution. Add on top of that the difference amount of cDNA used for PCR you have a lot of variables that affect the efficiency of your PCR.

However, it might be that you are using too much cDNA into your PCR, which can cause inhibition leading to late amplification that becomes less and less of factor the greater your dilution factor becomes. Thus narrowing your data points and producing a slope that is way below 3.3.

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Hi YuJ,
Since you are doing expression study, would't be more appropriate if you running reletive quanttification rather absolute quantification??
Just wander

Regards
Hadrian

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I think there is an inhibition in your PCR. 2 cases possible: You put too much template in your PCR or there is an inhibitor in your template (come from extraction). When you dilut your template you dilute the inhibition factor so the amplification is more efficient. That's why you have an efficiency >100%. To be sure of that, try with high dilutions of your template. The efficiency should decrease.

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I am having the same problem..i.e. high efficiency and I too think it is due to presence of an inhibitor which gets diluted out as I dilute my samples. All my dilutions have similar Ct values and in fact sometimes the more dilute sample gives a lower Ct value! Does anybody know to get rid of this inhibitor from the extract?
-Basu

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Basu- I am having the exact same problem. The advice I got from ABI was to purify my RNA samples with a column clean-up. So, I did the purification last Friday using the RNeasy MinElute Clean-up Kit. I got excellent RNA purity - I started with a purity of about 1.7 and ended up with purities over 2. However, I had large sample loss - about 50-80%. (Qiagen says I should recover 80-90% of my RNA samples with their columns, but so far I have not had good luck with recovery.) I am going to try RT PCR on these samples now and hopefully improve my efficiencies!

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I've used the Qiagen quick spin columns before I normally lose about half my sample. What the companys says and what actually happens can be two different things.

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I read this in an article called 'How Reliable is Your qPCR data?':

"When you're getting efficiency numbers greater than 100%, that usually indicates a saturation of the RT or the PCR, and you see less of a cycle threshold (Ct) difference between higher dilutions. That can sometimes throw off the slope on that high end and create artificially high efficiency. It is very important when you are looking at your standard curves to look for even spacing between all of them. If you do see compression on the upper end, throw out those points and recalculate the efficiency."

This leads me to a question about the dilution series that you normally run. I am wondering why the large range of dilutions (up to 1000x). If your qPCR is picking up the most dilute reactions in this case, it would seem that in your most concentrated reactions there could be way too much cDNA and produce the situation described above. I usually do dilutions in halves, e.g. 60 ng/ul, 30 ng/ul, 15 ng/ul...and do 6 dilutions. This has worked well for many HK genes as well as genes of interest.

Good luck!

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Hi All,

I am using total RNA to make dilutions for the standard curve, but having some difficulties to get good standard curve (efficiency is too high around 130%).
Would you suggest to use RNA to make the dilutions or this may cause any problem according to your experiences. thank you very much

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I assume your standard is a series of template with different concentration.
And real-time PCR works as you wish before.

The low PCR effeciency (<90%) usually means your primer is NOT good enough to produce realiable result.
A high PCR efficiency (>110%), on the other hand, usually means there is contamination especially PCR inhibitor.
Inhibitor can be explain your suspection of disuniform amplification; since higher concentration/volume of template you add into system (or your reaction well), it also bring more of those inhibitor as well. Thus in your standard reaction also have a gradient of inhibitor that have different inhibitor effect on its own.

One suprise real-time RT-PCR inhibitor took us a year to realized and get rid of, carbohydrate.
This thing most people will not consider an bad stuff, and it caused my previous real-time PCR with 190% efficient.

Good luck

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Calvino,

I do not do a 2 step qPCR. Instead, I do a separate rt-reaction. Then I make my serial dilutions for the standard curve. This works well for me and I have never done a 2-step qPCR.

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