negative control in qRT-PCR (View forum version)



Xiao Luo

Posted 13 January 2011 - 12:12 PM

Hello everyone,

New here and new for RT-PCR. I got some question about negative control.
Why no RT control still got Ct Value? I use SYBR green, if there are no ds DNA, it should not dye it, why still have signal form the icycler?
What about no template control, cause I did not run no template control, I do not know what kind of Ct data it would be?

Thank you!

Xiao Luo

Posted 13 January 2011 - 02:06 PM

why my baseline is not straight, it is like waves , does this affact my threshold line?

bob1

Posted 13 January 2011 - 02:12 PM

no RT control having signal indicates that there is some DNA in your RNA extraction. A no template control should always give you no signal - it contains no DNA apart from primers. If you have signal in this control, you have a contamination somewhere.

UBClabbie

Posted 13 January 2011 - 04:22 PM

there are several types of negative controls that you can use in qPCR.

Non-template control = no DNA, cDNA or RNA in reaction, amplification can indicate contamination of your reagents (ie. mastermix, primers) but this negative control is actually meant to detect primer dimer formation. if you get amplification, run your sample on a gel to see if its primer dimer (need to redesign) or if you're amplifying your target (ie. contamination)

A RT negative control is where you do not perform the reverse transcription reaction on your RNA sample = amplification indicates genomic DNA contamination. ie. your RNA extraction and isolation didn't go so well.... also could indicate general contamination of mastermix etc...


should note that avoiding amp. in your negative controls is sometimes difficult if you have been running qPCR or PCR a lot within the same day or time. Work away from cloning areas and clean your bench and equipment well before setting up reactions to minimize contamination. you can even setup reactions in a hood if you want to be super picky. sometimes I UV a hood to break down leftover DNA. Ct vales after 35 or 40 cycles is probably just contamination from the air.

Xiao Luo

Posted 14 January 2011 - 08:58 AM

there are several types of negative controls that you can use in qPCR.

Non-template control = no DNA, cDNA or RNA in reaction, amplification can indicate contamination of your reagents (ie. mastermix, primers) but this negative control is actually meant to detect primer dimer formation. if you get amplification, run your sample on a gel to see if its primer dimer (need to redesign) or if you're amplifying your target (ie. contamination)

A RT negative control is where you do not perform the reverse transcription reaction on your RNA sample = amplification indicates genomic DNA contamination. ie. your RNA extraction and isolation didn't go so well.... also could indicate general contamination of mastermix etc...


should note that avoiding amp. in your negative controls is sometimes difficult if you have been running qPCR or PCR a lot within the same day or time. Work away from cloning areas and clean your bench and equipment well before setting up reactions to minimize contamination. you can even setup reactions in a hood if you want to be super picky. sometimes I UV a hood to break down leftover DNA. Ct vales after 35 or 40 cycles is probably just contamination from the air.


Acturally, I only run RT-PCR three times, but every time the NO RT controls give me a Ct value higher than average Samples, but no more than 32, is that normal? Another confusion is that there are one set of data which NO RT control Ct value is 1 cycle smaller than sample or almost the same as the sample, how to explain this?( I run gel to confirm there are no DNA contamination in my RNA sample , then I run RT-PCR)
A little more about my experiment, I work on gene expression on bacteria RNA, I using total RNA extracted using Qiagen kits, and Dnase I (promega) treated to eliminate the DNA contamination, run pcr and gel confirm no DNA contamination, then run iCycler.
I attach some files about the result.
Thank you

  • Picture 1.png
  • Picture 2.png


UBClabbie

Posted 14 January 2011 - 10:05 AM


there are several types of negative controls that you can use in qPCR.

Non-template control = no DNA, cDNA or RNA in reaction, amplification can indicate contamination of your reagents (ie. mastermix, primers) but this negative control is actually meant to detect primer dimer formation. if you get amplification, run your sample on a gel to see if its primer dimer (need to redesign) or if you're amplifying your target (ie. contamination)

A RT negative control is where you do not perform the reverse transcription reaction on your RNA sample = amplification indicates genomic DNA contamination. ie. your RNA extraction and isolation didn't go so well.... also could indicate general contamination of mastermix etc...


should note that avoiding amp. in your negative controls is sometimes difficult if you have been running qPCR or PCR a lot within the same day or time. Work away from cloning areas and clean your bench and equipment well before setting up reactions to minimize contamination. you can even setup reactions in a hood if you want to be super picky. sometimes I UV a hood to break down leftover DNA. Ct vales after 35 or 40 cycles is probably just contamination from the air.


Acturally, I only run RT-PCR three times, but every time the NO RT controls give me a Ct value higher than average Samples, but no more than 32, is that normal? Another confusion is that there are one set of data which NO RT control Ct value is 1 cycle smaller than sample or almost the same as the sample, how to explain this?( I run gel to confirm there are no DNA contamination in my RNA sample , then I run RT-PCR)
A little more about my experiment, I work on gene expression on bacteria RNA, I using total RNA extracted using Qiagen kits, and Dnase I (promega) treated to eliminate the DNA contamination, run pcr and gel confirm no DNA contamination, then run iCycler.
I attach some files about the result.
Thank you



when it comes to qPCR, I'm not sure that running a gel is really a valid way to determine whether your sample is free of genomic DNA. qPCR is highly sensitive, much more so than running a gel. In fact, its one of the reasons that qPCR is used over other methods like traditional Northern or Southern Blotting. Just because you can't see it on the gel doesn't mean its not there.

qPCR is theoretically able to detect as low as a single copy, whereas gel imaging generally requires at least 0.5ug to see sufficiently detect.

As your Ct values from your RT are generally later than your samples, it looks as if you are probably having contamination issues. Either in your sample, your work area, or your reagents. If your RT is around the same Ct value as your sample then the signal from your sample is not valid, as all the signal could be purely from contamination. I suggest running another qPCR run and set up three reactions:

1. negative RT control
2. negative NTC control
3. sample

If your NTC amplifies, run the product on a gel to rule out primer dimer. If your Ct values for NTC and RT are around the same then its likely that your sample isn't contaminated.

Another way to determine sample quality is to use spec analysis on your extracted RNA. You want an Abs260/Abs280 around 1.8. I have used samples with as low a ratio of 1.45 and haven't had any issues. Ratio's around 2.0 indicate other sources of contamination. If you encounter this problem I suggest you look around in some of hte other subtopics in this forum for help on it.

Maybe replace all your reagents to rule out contamination there, and then setup your reaction in a biological safety cabinet or laminar flow hood. Work away from benches that involve cloning, running gels or any other types of experiments that use isolated DNA. I setup all my qPCR reactions in a separate room than where other experiments are run.

Hope this helps.

Side question: What do the two peaks in your melting curve correspond to? The sample vs. RT-control? If so, then you probably have primer-dimer or genomic DNA contamination issues....

Xiao Luo

Posted 14 January 2011 - 11:21 AM



there are several types of negative controls that you can use in qPCR.

Non-template control = no DNA, cDNA or RNA in reaction, amplification can indicate contamination of your reagents (ie. mastermix, primers) but this negative control is actually meant to detect primer dimer formation. if you get amplification, run your sample on a gel to see if its primer dimer (need to redesign) or if you're amplifying your target (ie. contamination)

A RT negative control is where you do not perform the reverse transcription reaction on your RNA sample = amplification indicates genomic DNA contamination. ie. your RNA extraction and isolation didn't go so well.... also could indicate general contamination of mastermix etc...


should note that avoiding amp. in your negative controls is sometimes difficult if you have been running qPCR or PCR a lot within the same day or time. Work away from cloning areas and clean your bench and equipment well before setting up reactions to minimize contamination. you can even setup reactions in a hood if you want to be super picky. sometimes I UV a hood to break down leftover DNA. Ct vales after 35 or 40 cycles is probably just contamination from the air.


Acturally, I only run RT-PCR three times, but every time the NO RT controls give me a Ct value higher than average Samples, but no more than 32, is that normal? Another confusion is that there are one set of data which NO RT control Ct value is 1 cycle smaller than sample or almost the same as the sample, how to explain this?( I run gel to confirm there are no DNA contamination in my RNA sample , then I run RT-PCR)
A little more about my experiment, I work on gene expression on bacteria RNA, I using total RNA extracted using Qiagen kits, and Dnase I (promega) treated to eliminate the DNA contamination, run pcr and gel confirm no DNA contamination, then run iCycler.
I attach some files about the result.
Thank you



when it comes to qPCR, I'm not sure that running a gel is really a valid way to determine whether your sample is free of genomic DNA. qPCR is highly sensitive, much more so than running a gel. In fact, its one of the reasons that qPCR is used over other methods like traditional Northern or Southern Blotting. Just because you can't see it on the gel doesn't mean its not there.

qPCR is theoretically able to detect as low as a single copy, whereas gel imaging generally requires at least 0.5ug to see sufficiently detect.

As your Ct values from your RT are generally later than your samples, it looks as if you are probably having contamination issues. Either in your sample, your work area, or your reagents. If your RT is around the same Ct value as your sample then the signal from your sample is not valid, as all the signal could be purely from contamination. I suggest running another qPCR run and set up three reactions:

1. negative RT control
2. negative NTC control
3. sample

If your NTC amplifies, run the product on a gel to rule out primer dimer. If your Ct values for NTC and RT are around the same then its likely that your sample isn't contaminated.

Another way to determine sample quality is to use spec analysis on your extracted RNA. You want an Abs260/Abs280 around 1.8. I have used samples with as low a ratio of 1.45 and haven't had any issues. Ratio's around 2.0 indicate other sources of contamination. If you encounter this problem I suggest you look around in some of hte other subtopics in this forum for help on it.

Maybe replace all your reagents to rule out contamination there, and then setup your reaction in a biological safety cabinet or laminar flow hood. Work away from benches that involve cloning, running gels or any other types of experiments that use isolated DNA. I setup all my qPCR reactions in a separate room than where other experiments are run.

Hope this helps.

Side question: What do the two peaks in your melting curve correspond to? The sample vs. RT-control? If so, then you probably have primer-dimer or genomic DNA contamination issues....


the two peaks are the reference gene vs. gene of interested, maybe as you said, I will try to run another set with no rt control and no template control, and also move my work place to the hood try to avoid the DNA contamination.
Still , you answer help a lot to me a new guy in RT-PCR, I still working on reading materials, so hope those can help me know better, thanks a lot for your answer, nice to talk to you. Hope we can communicate more about RT-PCR.
Thank you