I am trying to quantify a cytokine gene (for example, TNFa) expression when the cell is infected with a virus. I have used RNeasy RNA extraction kit to extract RNA and a DNase treatment step has be included. The Ct in RT(-) tube is around 25, while Ct in RT(+) tube is around 22. It seems that TNFa gene is not expressed much. In this case, how can I use the Ct from RT(-) adjust the Ct of RT(+) to get a real figure which is contributed from mRNA? Thank you.
I am not sure that it is technically valid to do what you want. However, there is another way to estimate and correct for the amount of gDNA you have in your samples, see the attached link.
Is it possible to design your primers to span introns?
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326333/
I am not sure that it is technically valid to do what you want. However, there is another way to estimate and correct for the amount of gDNA you have in your samples, see the attached link.
Is it possible to design your primers to span introns?
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326333/
Dear Bixing,
At what cycle does your NTC amplify? What is the RNA quality? What reference genes are you using and what does their amplification look like?
From your data, you cannot conclude anything as quite obviously either
1) at least 10% of your amplification is from genomic DNA contamination (your DNase digest wasn't working). 10% because a delta Ct of 3.3 equals a 10-fold difference in expression. I accept data that has at least 6.6 Cts difference between -RT and +RT samples (less than 1% contribution of genomic DNA to the PCR amplification), but generally, -RTs should not amplify properly. As Anil says, do your primers span exon/exon borders?
Could also be that your amplification is actually due to primer dimer, so please let us know what your melting curve looks like and what the PCR product(s) looks like on a gel.
or
2) there is contamination in your reagents/PCR preparation (this is why I am asking about the NTC).
In both cases, your results are simply due to an artefact of your experimental set-up and should therefore not be used in any way.
What it means is that you need to repeat your RT-qPCR.
I've attached the MIQE guideline paper to help you with clarifying on how to get good and valid RT-qPCR data.
A CT of 25 for (-)RT is far too low. It should be >30, better >35, ideally you would not get any amplification form the (-)RT control.
As Petra pointed out, a NTC would be helpful to see if this is really an amplification of genomic DNA (in which case you should optimize the DNAse treatment, or design primers that would not amplify well from genomic DNA [->intron-spanning]) or if you have a contamination (then either burn the lab down or start over with new primers).
Under no circumstances I would trust a qPCR where the controls that are expected to be actually negative give CT values of around 25 (or
A simplistic approach to the equation you may be looking for would be:
gDNA contribution factor = EAMP(Cq of sample - Cq of -RT rxn)
E.g., the resulting number would represent the fraction of your [calculated relative quantity] signal that came from gDNA contribution (assuming that the extent of gDNA contribution to signal is constant across all samples if not running a -RTrxn for all targets for all samples).
But - everything the others have stated above far outweighs the utility of such an equation. However, with -RTrxn Cq values of (as Jochen points out above) >30 or (better) >35, this equation seems to prove helpful...
In other words, at 100% efficiency (where EAMP = 2), with a sample (+RTxn) Cq of 22 and a -RTrxn Cq of 25, the equation above indicates that 12.5% of your signal is coming from gDNA (in theory, and in the best of all possible worlds, that is).
Dear Bixing Huang
I want to tell you that technically your experiment is working because you observed the 3 Ct difference between the (-) and (+) that means your gene of interest is upregulating in the (+) samples. Please explain that is (-) Rt a sample without virus infection. If yes then is your gene have a basal expression that is upregulating in presence of virus.
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