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Help with Real Time PCR Well To Well Variation - (Mar/15/2009 )

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I am new to real time pcr. I am finding that my well to well variation for technical replicates is on the high side. Sometimes it is greater than 2 CTs. I am using 96 well plates with optical caps so I do not think evaporation is causing this issue. I have recalibrated my pipets several times as well. Could this be an instrument issue? It seems to be random and not an edge effect.

-babylabrat-

This issue is mostly caused by instrumental contamination. Your PCR machine needs cleanup and calibration.

-pcrman-

I thought so as well but the machine is still new it is a 7500. My labmates do not have this issue. I wonder if it is assay specific or something I am doing wrong.

-babylabrat-

Assuming that your pipettes are calibrated, the instrument is clean (when you take a look at the block using the instrument's optical system you do not see any bright spots), and you have a good assay, I am going to guess that maybe you are not pipetting your solutions well? I always reverse pipette the qPCR master mix since even at 1x it is viscous. Also, none of my volumes is less than 2 microliters. Since you are doing technical replicates hopefully you are preparing the whole reaction and then pipetting the whole thing into each well. If you are adding the master mix first and then the template to each well you may be having issues with pipetting small volumes.

-ivanbio-

ivanbio on Mar 16 2009, 11:55 PM said:

Assuming that your pipettes are calibrated, the instrument is clean (when you take a look at the block using the instrument's optical system you do not see any bright spots), and you have a good assay, I am going to guess that maybe you are not pipetting your solutions well? I always reverse pipette the qPCR master mix since even at 1x it is viscous. Also, none of my volumes is less than 2 microliters. Since you are doing technical replicates hopefully you are preparing the whole reaction and then pipetting the whole thing into each well. If you are adding the master mix first and then the template to each well you may be having issues with pipetting small volumes.


My painful experiences in real-time PCR totally and absolutely support ivanbio's points. Try to "preparing the whole reaction and then pipetting the whole thing into each well", this is very important to eliminate well variation. And other suggestions are:
1. Whenever you aliquoted any solution, make sure to mix them beforehand.
2. When you pipette your solution, make your pipetting same each time. I mean if you pipette all solution in tips out for the fisrt well, then the same for the rest two and if you leave a little solution (around 0.5-1.5 microliters for 100 microliters tips, caused by the pipette) for the first, then the same for the rest two.

In my lab, we prepare qPCR mix by ourself not commerical qPCR master mix. As long as we keep above points in mind, we could eliminate well to well variation. May it help you and good luck.

-WOW-

Maybe you don't mix your cDNA well enough before transferring to the plate? Eg. pipetting up and down 5Ál in a total volume of 40Ál is not sufficient. In my experience this has the biggest effect on the replicate precision.
Currently, I have well variations on a plate within ~0.1 Ct when pipetting with single channel pipette (with 8channel its not that accurate but also ok.) and I'm adding master mix and cDNA separately. Maybe also your reverse transcription reagents "disturb" your reactions?
Well variations of more than 1 Ct are unlikely caused by imprecise pipetting. this assumes you put in one well > double amount of template compared to the other well.

-tea-test-

Thanks for the good info. One of my colleagues suggested I should spin down my plates first. I guess I was not as careful pipeting into the plate as I thought I was. After doing this my replicates got a lot better. Plus I started to use mini master mixes and reverse pipet technique. Now I only wish the plate centrifuge was not as far away.

-babylabrat-

carefully seal the plate between each row / line and spin down the plate. Bubbles at the bottom of the wells are no problem in my experiments....

-markusda-

Using a mini mastermix, switching pipet tips every well and spinning down the plate before QPCR solved my issues. I even found a cool product to spin down the plates at my bench since the table top is in another lab. It is called the HandyFuge. Basically it is modified salad spinner which can also spin down pcr tubes. Check out the website: http://www.handyfuge.com

-babylabrat-

not sure if people are still paying attention to this thread: but any further thoughts on the necessity of spinning down plates before putting in the thermocycler. A post-doc in my lab has reasoned that it doesn't really matter with her hotstart assay because the heat will disrupt the bubbles and cause the solution to evaporate and then condense regardless. Is this faulty reasoning?

-dtae-
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