RT control positive!

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cubita1
cubita1's picture
RT control positive!

Hi,
I am using Sybr Green for my qPCR. I already have the optimal conditions for the assays (T and primer concentration).  I am using the amount of cDNA corresponding to 150ng of total RNA because the gene of  interest has very low expression.
The negative control (NTC) is Ok. However  I have fluorescence signal in RT control, similar to the signal for cDNA. We thought it was genomic DNA and we used 2 methods to fix the problem. We used DNAse and also some kit  from Qiagen to eliminate gDNA. After the treatments the problem is the same.
If I dont have cDNA, or gDNA in my RT control, then, what is the template for the polimerase?? The RNA?? Maybe I have to use RNAse after cDNA synthesis.
I would like to know If somebody has some suggestion. I am thinking that there is some kind of science fiction in my tubes...It is not contamination because my NTC are clean..
 

Ivan Delgado
Ivan Delgado's picture
 

 
Hi cubita1,
Is it possible that your RT control signal is coming from primer dimers? Typically you would see primer dimers in the NTC too, so if you do not see it then likely this is not the case for your RT control. Could you explain in more details what you mean by "signal" in your RT control. What is the difference in Ct value between your RT control and your other samples?
 

cubita1
cubita1's picture
Hi Ivan,

Hi Ivan,
The Ct for RT control is always around 28-30. One of my genes has the same Ct. For other genes, the Ct for the samples is 20.
NTC are always clean, at least after 35 cycles.
Thanks

Ivan Delgado
Ivan Delgado's picture
 

 
In all honesty a Ct value of 28-30 for a RT control is very high. If the amplification curves for your RT control look real (sigmoidal), then there is little doubt that there is some kind of contamination. By any chance are you amplifying bacterial RNA? If so, many qPCR master mixes are made in bacteria so it is possible that the source of your contamination was in your reagents to begin with. 
Beyond that I can't really think of anything else based on the information you have provided. Maybe if you give me more details I can identify another source for the problem. 
 

cubita1
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Then, do you think that a Ct

Then, do you think that a Ct of 32 is normal for RT control? It dosent have a nice sigmoidal shape but in the meltig curve is under the sample with cDNA. My sample with cDNA has a Ct of 25
Thanks
 

Ivan Delgado
Ivan Delgado's picture
 

 
One of the "unspoken" rules of thumb in qPCR is that as long as your negative control is 10 Cts later than your lowest sample signal, you are ok. Ten Cts is equivalent to a 1,000-fold difference, so if you are quantifying one microgram of RNA, this would be like saying that you have a background noise of one nanogram (a 0.001% error). For all intended purposes, more than acceptable.
In your case, the Ct difference is only 7. So while it is still a big difference, it is one order of magnitude smaller (100-fold). You could argue that this difference is still big enough for the error to be insignificant as far as the quantification of your samples is concerned, but unfortunately since we are dealing with PCR a Ct difference of 7 is for the most part considered too small and the error too high. 
Hope this helped. 
 

qyang_2000
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I run into almost same

I run into almost same trouble as yours. I tested 7 genes with one negative RT control (no transcriptase) in real time PCR. Six out of 7 showed the same size bands as my target genes, respectively. The negative RT control never had nice sigmoidal shape (not at all in most of them) but the disassociation curves are under the sample with cDNA. The sets of Ct from cDNA samples and negative RT controls are as follows: (16, 30), (17, 19), (19, 32), (20, 29), (23, 26), (24, 31), and (26, 36 -- no band from negative RT shown on the gel). 
Can anyone has good explination or suggestion?  Thanks in advance.