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How & where to prepare qPCR


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#1 goyabean

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Posted 10 August 2009 - 09:15 AM

There are a lot of discussions about using separate rooms for preparing your qPCR reactions but I am still unsure of the *best* way to prepare your qPCR reactions in order to eliminate (to the best of our ability) contamination from the environment.

What is your procedure for preparing the qPCR? When do you switch rooms?

My general ideas right now are:

1. Prepare common mix in some *very clean* room (ex. a laminar flow hood, after all things UV light treated)
2. Possible UV light treat your common mixture with tube open in laminar flow hood
3. Aliquot your common mix into tubes.
4. Prepare your "No Template Controls" (NTCs) in the same hood & place in plate
5. Prepare your standard cure and unknown samples out in some "dirty room"
6. ???

Here's my question...where do you prepare your positives and samples? Do you bring the DNA into the laminar flow hood? Or do you take the CM tubes and plate out to the same area where your DNA is? Do you need a third *intermediate* room?

Please post suggestions. Thanks!

#2 ivanbio

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Posted 10 August 2009 - 10:07 AM

I too have come across this question before and these are my comments:

1. If the point of your NTCs is to serve as controls against contamination, why would you prepare them in a separate room? Doesn't that essentially defeat the purpose of an NTC? For example, lets say you are running 10 samples and you get cross contamination from sample 2 into all the other 9 samples. If the NTC was prepared in a different room, this cross-contamination would not show up in the NTC so there would be no way of knowing that you got cross-contamination. Yet, if the NTC was prepared together with the other 10 samples, you would catch this cross-contamination in the NTC and as a result question the results obtained in all 10 samples. It seems to me, based on this rationale alone, that preparing the NTC in a separate room is not a good idea: it would make the NTC look good, but it would only test for contamination already present in your reaction mix, not any cross-contamination that occurs later on in the experimental setup.

2. I am not aware of any study showing that UV treatment is any better than a good wiping of your work area with a 70% ethanol solution and/or bleach. Actually, in my experience UV light barely gets rid of even very low levels of DNA contamination, so if you have a little more than a minor contamination UV treatment will simply not make a difference. Plus UV light, at least the amount you would need to use for half-decent clean-up (at least 1 to 2 hours), is going to go to town on your plastics. Be ready to replace your pipettes more often.

3. Moving your sample from one room to another, if not done properly, can cause more contamination than it prevents. The assumption that you have separate rooms for qPCR implies that these rooms are contiguous. If you have to leave one room and enter a hallway before you can reach the next room, you are for all intended purposes defeating the purpose of having room separation for qPCR setup.

Just my opinion. If you feel like you need to have separate rooms to set up qPCR reactions, consider testing if the separation is really making your workflow better or worse. If should be pretty straight forward to devise an experiment to test for contamination and run it using both workflows. If you get no difference between them, it would seem to me that there is little point in adding all this extra work for something that really does not solve any problems.

Many people that implement separate rooms for qPCR setup do not realize that the only reason why we talk about this is because the EPA published a guidance document back in 2004 for the testing of environmental samples by PCR (follow this link). If your are testing samples that have truly tiny amounts of DNA present (environmental samples), then it makes sense to implement this separation because it is critical that your background level of detection be at its lowest possible. Yet, for most qPCR applications this level of control is overkill. Remember: statistically speaking a qPCR assay cannot detect less than 3 copies of your template of interest (see MIQE guidelines), and most qPCR assays are really not designed or optimized to detect less than 10 copies. The kind of background you will be able to eliminate with room separation is in the order of a copy or two, if that.

Ivan
Carlsbad, CA

#3 cellcounter

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Posted 10 August 2009 - 10:59 AM

There are a lot of discussions about using separate rooms for preparing your qPCR reactions but I am still unsure of the *best* way to prepare your qPCR reactions in order to eliminate (to the best of our ability) contamination from the environment.

What is your procedure for preparing the qPCR? When do you switch rooms?

My general ideas right now are:

1. Prepare common mix in some *very clean* room (ex. a laminar flow hood, after all things UV light treated)
2. Possible UV light treat your common mixture with tube open in laminar flow hood
3. Aliquot your common mix into tubes.
4. Prepare your "No Template Controls" (NTCs) in the same hood & place in plate
5. Prepare your standard cure and unknown samples out in some "dirty room"
6. ???

Here's my question...where do you prepare your positives and samples? Do you bring the DNA into the laminar flow hood? Or do you take the CM tubes and plate out to the same area where your DNA is? Do you need a third *intermediate* room?

Please post suggestions. Thanks!

We have been doing qPCRs in a "dirty" room, without any contamination. Negative controls do not amplify anything ever.

So, while good measures are always good, I would not get myself bogged down in intricate planning unless..

1. You are dealing with a project that requires GMP.
2. Your negative controls are giving some positive results.
3. You are dealing with an environmentally abundant target.

Just do the experiment with reasonable measures to avoid contamination, and see if you really need anything more.

#4 goyabean

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Posted 11 August 2009 - 07:11 AM

Thanks for your view points. I have also thought that it was overkill to worry about different rooms and UV light and all that, until I can across this new assay and I can not get the NTC to decrease from Ct 29. My most recent experiment was with bleached and UV exposed pipettors, and 0.2 uM filtered water, new master Mix, bleached and UV'ed laminar flow hood, on top of that--I didn't even take the positive DNA out of the freezer. I still got a Ct of 29 so I guess that is just the background of my assay. I doesn't make sense because the background should be lower but I get the same in the "dirty room" versus the extra clean conditions.

I will be taking the precaution of changing my primers and probes to a whole new stock but I got this signal the very first time out of the box with this set.

Thanks all for the discussion.

#5 ivanbio

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Posted 11 August 2009 - 07:50 AM

If you get a Ct of 29 on your NTC, there is a good chance that it is not contamination but something else. My guess would be that you have primer dimers or non-specific amplification. Looking at the Melt profile of the NTC may give you some clues about this. If possible, my suggestion would be to re-design the assay. In my experience when you get this kind of background signal on the NTC there is almost no chance to get rid of it.

Ivan
Carlsbad, CA

#6 susanna

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Posted 10 February 2010 - 08:06 AM

there probable is contamination in your primermix

If you get a Ct of 29 on your NTC, there is a good chance that it is not contamination but something else. My guess would be that you have primer dimers or non-specific amplification. Looking at the Melt profile of the NTC may give you some clues about this. If possible, my suggestion would be to re-design the assay. In my experience when you get this kind of background signal on the NTC there is almost no chance to get rid of it.



#7 Microbes in action

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Posted 24 February 2011 - 08:37 PM

I agree with all the above discussions.

With reference to clean experiment set up, jus try to make an experimental set up, as clean as possible, try to follow all the basic aseptic procedures, and clean pipetting procedures. Changing the tips, Changing the gloves, and not moving the tips over the open reaction tubes. Having clean rooms for each and every step is an overkill and can be adviced for GLP's and regulatory works.

Apart from this if you would want to identify the product formed in your assay, try to include dissociation curve in each and every run you make and try to estimate the melting temperature of each product and also the derivative of the product formed. This can clearly give you the information of the product formed.

Also check if the dissociation curve NTC and the product are the same.

Hope this also helps.

Cheers.

#8 phage434

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Posted 01 March 2011 - 02:05 PM

Avoid autoclaving reagents and plasticware. Use filter tips. These are much more important than a separate room. The less done to your water, the better. Think about reagent quality water, which you then don't do anything to. Or use a master mix, so you don't need to add water. Master mixes are good simply for reducing the number of potential sources of contamination, and in standardizing conditions.

#9 dtae

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Posted 06 March 2011 - 12:54 PM

ivanbio, I think you're incorrect about the roots of separate rooms for PCR having its roots in the 2004 EPA report. I've worked in multiple labs established before 2004 which had separate rooms, positive pressure and other such techniques in an effort to eliminate contamination.

I've personally worked in several labs with very high levels of measures taken to eliminate contamination (separate rooms, positive pressure, frequent UV and bleaching) and labs with little to no such measures. I've had little trouble with contamination and haven't noticed a difference between labs.

Those interested in the topic should also see:
http://www.protocol-...h__1#entry40274

#10 biznatch

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Posted 29 May 2012 - 06:21 AM

My lab does tons of PCR and qPCR. Everyone sets their PCR up at their own lab bench that they use for all their other work. No special cleaning, no special pipettes, no problems with contamination. We use filter tips for anything PCR related, and use Milli-Q water. I aliquot my Milli-Q water in 1.5 mL tubes so that any given tube of water is only used for a few PCR's. If I use the water from say a 50 mL tube, I usually start seeing contamination after 10 or 20 PCR's.




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