If the CT changes with the concentration of dimer, it is not primer dimers but background amplification from environmental contaminants. In either case, 37 cycles usually pretty safe to say you have no legitimate targets in your system. I would not be overly concerned as long as my real samples have Ct at least 5-10 cycles less. If you are still concerned, do a dilution serial on your sample to see when it becomes non-linear. As long as your sample still in the linear range, you should be fine.

OK - the great advantage of SYBR is that you can look at the melting curve of the product. Examine the melting curves of your "negative" samples with those where you know the virus is present. The true positive samples should have a single, clearly defined peak-like melting curve. Now, consider the melting curves of your artefactual signals. If it is comparable to the true positive in terms of melting point and tightness of the peak, then it is most likely that you have gotten some virus into your buffers or primers etc - confirm by cloning and sequencing the product. If it is different (temp, width), then your primers are cross-reacting with some DNA that has made its way into your system. If it is systemic then this second peak may also appear in your true positives in a given run.

Solve the immediate problem by discarding all your working stocks - enzyme, primers, buffer, and if is present when you redo the assay with fresh aliquots of primers (you do aliquot your primers, right?) then throw all your primers away.

Once you've done that - re-examine your primers for cross-reactivity with human DNA and redesign if necessary, and order fresh.

Good luck

G

Hi

I also agree with the suggestions above by Dr. Gallagher above...don't go alone by the Ct values also chect the melting curves of the product to assess their presence...you can also use Agarose/page gels to ascertain the expected product and relate it the the melt profile initiially

if the product is coming even in the blanks or controls...it indicates contamination....so do the anylysis carefully...

Always go with melting curve analysis for SYBR green as Dr Gallagher mentioned. Interchange reagents one by one to detect for any possible contamination. If you observed single peak for samples in melting curve analysis and no band in agarose gel, avoid ntc peak observed after 35 cycles. Some qPCR platform like eppendorf has flexiblity of adjusting your Ct values.

This is usually primer dimer in my experience with SYBR green.

If the CT changes with the concentration of dimer, it is not primer dimers but background amplification from environmental contaminants. In either case, 37 cycles usually pretty safe to say you have no legitimate targets in your system. I would not be overly concerned as long as my real samples have Ct at least 5-10 cycles less. If you are still concerned, do a dilution serial on your sample to see when it becomes non-linear. As long as your sample still in the linear range, you should be fine.

I endorse Grants comments. I would also like to add that a lot of people who do real-time PCR often do not realise to potential for cross-contamination of samples and PCR reagents with previously amplified products / plasmid standards etc. Generally speaking if you are going to do qPCR it is vital that you have separate dedicated pipettes for different stages - one set that only ever see PCR reagents ( no copy number), one set for unamplified samples (low copy number), one set for plasmid standards and amplified products (high copy number) and where possible you segregate the preparation steps eg RNA extraction done in a hood, aliquotting of reagents in another. Never use the same pipettes for plasmid standards or amplified products to aliquot reagents - even if you use filter tips. I try and UV irradiate my pipettes before and after setting up a run in a UV irradiator. If you have a hood with a UV light you can use that for sample prep. There is not much point in repeatedly making fresh primers etc if the primary source of contamination is not dealt with. You can reduce the presence of contaminating pre-amplified product by using the dUNG approach or adding artic shrimp DNAse (for cDNA - it is ds DNA specific)). If doing a DNA PCR, the artic shrimp DNAse has to be inactivated before adding target DNA.

The other question which arises - is there an "acceptable" level of "contamination" in qPCR ?. Ideally one would like zero all the time, but often we have no choice but to perform all our functions on a lab bench of a few sq mtre. If your target amplified product gives you 1 million copies and the NTC gives 10, then is it materially going to make a difference to your results provided you stipulate that the background / threshold of detection is 10 copies?. If your target gives you 20 copies, then obviously a NTC of 10 is of concern and needs to be repeated. If your assay is a diagnostic Yes/No, then no question that a "background amplification" is unacceptable. One of the beauties / curses of real time PCR is that it enables you to visualise these issues. If you stopped your PCR at cycle 35, would the result be any less/more valid??

Must stop ranting....

Hi

I also agree several concerns raised by Ian......real-time PCR is highly senstive and this sensitivity also detects contamination easily....so be carefull in planning, exceuting the experiments...and analysis of results..

bye

Hi,

If your total number of cycles is 38 and your NTC has given a Ct value of 37 the, you need not worry about that, provided your sample has already amplified within 25-32 cycles or earlier. Secondly, I would suggest you to make fresh stocks of primers and keep the SyBr mix as aliquots to prevent cross-contamination from other users. Also, be very sure of the water that you are using. Most of the results of negative control come from contaminated water sources.

Best!

To add to my rant - a question all PCR users should be able to answer:

The Govt of Utopia has embarked on a screening programme for the single copy "Foot In Mouth" gene which has a 50% prevalence (- mostly but not exclusively men and not to be confused with Foot and Mouth Disease). They plan to take buccal swabs (estimated 100 cells /swab) and do 40 rounds of PCR per sample. Their diagnostic lab can screen 100 people /day. How many people or how many PCRs have to be screened in order to generate the equivalent number of gene copies of Foot In Mouth as found in a whole human being??

We can currently amplify DNA from forensic samples from decades ago. Diagnostic labs can deal with 100s of samples per day. The incidences of cross contamination are a function of the number of samples and amplification products. Whilst they are mercifully small in a well run lab, it will become increasingly difficult over time to maintain and "clean facilities" which will be needed say in the event of a pandemic. We are potentially creating the equivalent of "PCR space junk" which may impact on the future viability of a valuable diagnostic technique unless people are made very conversant as to how to do clean PCRs and decontaminate / inactivate potential cross contaminants. I suspect swabs of most benches in a standard molecular biology lab will be PCR positive for a plasmid vector.

Check your water, primers and pipettes with aerosol contamination

Hi

In addition to the above possible reasons , I would incline to know about the brand of your instrument , some of the instruments goes faulty simply because it needs to be re calibrated , so if all the above mentioned reasons have been excluded think in this possibility.

Hi Mohamed,

I am curious, were you able to determine the source of amplification? I still believe the amplification you see in your NTC at high cycle number is due to primer dimer. This is somewhat common using SYBR chemistry. Also, since you are investigating influenza, there is an increased probability of palindromic sequence and hence primer dimer. Just my two cents, I have seen this many times.

Hi there,

Maybe you have a primer dimer problem. Sometimes I have it too, but after checking the melting curve you realize it is not contamination but primer dimerization. Maybe you should re-design your primers. Can you see these products on a gel too?. Try to increase the annealing temperature to avoid dimers.

I hope this help.

Best regards,

I do not think it is a primer dimer problem. It is just a background noise, often seen with q-PCR techniques and should be ignored.

what is the Tm for your "primer dimer"?

The sixty four dollar question from Dr Codony...primer dimers usually come in at a Tm of around 70C. If PCR product is a significantly higher melt, then the PCR is at least clean. The question is how much of the fluorescence is due to the primer dimers? If they are priming the template, then fine, minimal primer dimers - but if no template, as in the case of NTC, primers are all dressed up and nowhere to go, so they gather together and wreak their mayhem. A Ct of around 37 for a NTC is not worth worrying about...at that number of cycles, there is going to be noise/primer dimers. Don't sweat it. If it's a real issue, obtain fluorescence at a higher temp -above the primer dimer melt, but just below specific product. Fluorescence INTENSITY is lower at the higher temperature, but the Ct values if there is no dimer interference, will be the same.

Pl change water and check pipetts

If this primer dimer, can I proceede in the experiment or it will affect ؟ how to prevent it

That would depend on the Ct's for your unknown. If they are at or near the signal in your NTC (you mentioned ~37cycles), then I would try a pre-amp step to increase your initial unknown cDNA concentrations. This would lower your Cts and the signal in the NTC will have no significance.

Has anyone considered the Microgene Polymerization Reaction (MPR) as the cause?

See the following articles - all by Itsko et al.:

1. Exposing cryptic antibacterial activity in Cyt1Ca from Bacillus thuringiensis israelensis by gnetic manipulations. FEBS Letts 581: 1775-1782, 2007

2.nitiation of the Microgene Polymerization Reaction with non-repetitive homo-duplexes. Biochem Biophys Res Commun 368: 606-613, 2008

3. Thermodynamics of unstable DNA structures from kinetics of the Microgene PCR. J phys Chem B112: 13149-13156, 2008

4. Kinetics of repeat propagation in the Microgene Polymerization Reaction. Biophys J 96: 1866-1874, 2009

5. Tandem repeats in a new toxin gene from Bacillus thuringiensis and in other cry-like genes. J molec Microbiol Biotechnol 20: 204-210, 2011

6. Tandem DNA Repeats: Generation and Propagation in the Microgene Polymerization Reaction and in vivo. Chapter 6 (pp. 175-202) in, Applications of Thermodynamics to Biological and Material Science, ISBN 978-953-307-980-6 (Ed. Tadashi, M). InTech Publishers, 2011

Best of Luck!

Could you mentioned how to include an amplification reaction before qRT-PCR in details

Hi Mohamed,

Are you working with low initial total mRNA concentrations? If so, you may want to try a higher yield isolation method. This in combination with a pre-amplification step may allow you to see amplification at lower cycle number.

A number of companies have kits specifically for this. These are ideal if you have no experience optimizing cycle parameters on your own. A publication search (or Google search) would be most helpful in this case.

Hi Mohamed,

In addition to what has already been said, I would recommend to use a decontaminating solution to regularly clean your pipets, as well as the working area of your bench, especially if you are not using a PCR-hood. Doing so before and after each reaction set up I have been able to get rid of positive NTCs.

Hope this helps.

Hi Mohamed,

NTC amplifications can be a problem with SYBR reactions as SYBR is non-specific and binds to any dsDNA formed. Amplification of the NTC is usually caused by contaminants in your reagents or in the environment. The easiest (and probably cheapest) thing to do is replace all of your PCR reagents, aliquot new reagents and use new PCR tubes, filter tips etc.

Other suggestions are to separate your RNA isolation, cDNA synthesis and PCR reactions between different labs as much as possible to prevent cross-contamination. Also be very carefull with your post amplification products as these can contaminate your future reactions! I also run a melting curve with all my qPCR reactions, this can indicate if you are getting primer-dimers in your NTC.

In practice though if your NTC is passing threshold >35 and your gene of interest is >10 cycles lower, this is usually acceptable as the background 'noise' will have a negligible effect on the signal from your gene. Good luck with your PCR

Interesting potential answer Arieh. I have often wondered why I have occasionally got a smear in some PCRs that was obviously not a primer dimer but a concatermeric construct of some sort, but not pursued it further. Worth going back and re-reading the Shiba et al PNAS paper 1997.

Finally, someone who is 'in'!

We are not pursuing this technique further, Ian, but believe that it can be useful in the field of protein engineering.

It can be fun if we find a way to cooperate on this issue--perhaps by exploiting funds from the EU; we shall be delighted to.

You may correspond to my email address [email protected]

hi,,

I agree with sir, Dr. Gallagher suggestions..

It may be the environmental contamination and dilution problem of ur primer and gene..

First check ur primer is specific or not.. by using primer blast .. may be it can give the amplification in ur non infected sample..

separate the process of RNA isolation and cDNA synthesis and purify it using DNAse treatment after RNA isolation as suggested by Michael Rotherham ·

Then dilute the primer and gene u used before that make it clear that ur stock is not contaminated , use fresh stock for real time.. make sure ur SYBR green also not contaminated.....(May be virus contamination is present in stock of non infected samples)

Check the melting curve in real time that primer dimer is formed or not in both samples.

Gud luck...

Hi Mohammed,

Are you filtering your mq water used for dilutions, secondly as long as your NO cDNA samples are 8-10 cycles apart than it may not be affecting your results.

Good luck

Shadma

Hi every one,

as I have experienced this before, I suggest that because contamination in NTC can happen easily by using especially contaminated DEPC water or lab area, you can ignore it if it is few cycles like 5-10 cycle further than your sample... Good luck

Hello,

To see if this is caused by some contamination or inespecific dimers of your primers, you should do you PCR with only water (or with what you dilute your samples) from a "new" botle and with the bottle you used.

If you have in both you it is caused by inespecifc dimers of your primers, so you should do new ones.

If do not have with any, you have a contamination in other reagent.

If you have on the used bottle and not in the new, well you found it.

Good luck.

Hi Mohammed,

Is your problem resolved.