Inconsistent results from replicates in Taqman real-time PCR - (Jun/24/2006 )
I have a similar problem as madeinspain.
I do triplicate std curve with a master mix but add cDNA separately for each triplicate. I don't vortex my cDNA though. But I mix by pipetting gently. I use Qiagen SYBR Green. Machine: Corbett RG-3000. Cts below 30 but difference between each Ct of each triplicate is about 4 Ct values. Some triplicates have Cts running all over the place, eg Ct1 = 15, Ct2 = 28,Ct3 = 10. All for actin gene. I can't get an average efficiency value like this. My efficiencies range from <1.0 to >2. What's wrong?
copy numbers at about 10 copy numbers already suffer from statistical variations. The poisson's standard deviation of 10 is sqrt(10)=3.16 . More below 10 its even bigger than this rule. And at very low copy numbers the LightCycler is also affected from unspecific DNA binding in the glass capillary. So samples, containing very low one-digit copy numbers can get enormous CP-values (but the specific product).
We are also working with very low copy numbers and are satisfied to get standard deviations of 0.5-1.0 . (If efficiency would be two, than a SD of 1 means that the template number varies from the factor two). If your high CP -values are results of the low copy numbers, your bacterial DNA must be very low concentrated. Inhibitors in a sample don't affect the SD so much (in my experience). Another Quote: I don't know how the Mobio kit works, but if it's based on a magnetic bead extraction, than you must take care of a magnetic separation. At low copy numbers (below 50) magnetic beads in real-time PCR lead to very high stantard deviations. Perhaps this could be an explanation of your 4°C experiment (very low concentrated DNA binds to magnetic beads. These concentrate the DNA and are inserted in PCR, what leads to a specific product).
Just some experience from my work
My suggestion is increasing amount of template!
Based on your target, bacteria DNA in human blood samples, and your result of high Ct value (>35), I suspect that your target DNA concentration is very low in PCR reaction, such as <10 copy per reaction, even under 1 copy per reaction. If that were true, it will be impossible to obtain a good amplification with low variation.
My experience with RT-PCR using SBRY green system, if the Ct value is above 37, and PCR amplification efficiency is normal (near 2), then I might obtained CT value with high variation, which I believe it results in low copy number of target in reaction.
Increasing template amount might help if the target copy number have been increased at least above to 10 pre reaction…
at 4C - check pH - freezing at -80 C - overnight helps - sonication helps to shift to lower Ct
but after that do not QUANTIFY - sonicated vs nonsonicated
shurelly copy number will be the same but Ct can be STRONGLY different
taqman or hybprobes ? as I remember taqmans originally were not reccomended for
lightcycler by inventors of this instrument smile.gif 15 Taqs from different companies give dif results (see
PubMed) may be it is because 5' nuclease domain of Taq is thermolabile ( see PubMed)
hybprobes can work nice with Stoffel or KlenTaq ( see PubMEd)
My collegue had the same problem, it is necessary to digest blood cells prior to extraction of bacterial DNA. please look up for publication of Handschur et al.
I hope U find this necessary. Did U already try to use a purification kit after DNA extraction?
First,thank you very much for your reply.
I really have a problem because I meant that I make one tube and split it 3 ways to get my replicates
I am using my self-designed primers and protocol to amplify bacteria DNA, no RNA. It is very specific because when I run the product of the capillaries where I have got a signal into an agarose gel I just see one band corresponding with the bacteria amplicon. It seems that the human genomic DNA interference with the bacteria DNA or something related with the DNA extraction procedure... Oh.....it´s quite fustrating!!
Another thing I have seen is that when I perform the real time PCR the same day I have isolated the DNA (with Mobio kit) I get no signal!!If I repeat the PCR with the same eluted DNA after two days at 4ºC some replicates start giving signal(???).
I use the LightCycler MasterPlus Hybprobes (Roche). I can give you more detalis if you want.
Hi I read your discussion. I have similar problem. I amplify 18S RNA: I add the same mix and template separetly to three tubes and for exaplme 2 of them are have very close Ct and one is about 4- 5 cycles different. No idea. Service says that files look ok that means corbett equipment is working ok. Any idea what can I do. I use 94C 10s 56C 20s 72C 20s (very difficult to beleive that this is pipeting error. I pipete 7,5 microlters of mix and 7,5 of template with primers.
how's your RNA quality? what sort of 260/280s are you getting?
I worked with the LightCycler for a long time, currently I working more with ABI systems. However, I can tell you that a lot of the variation that you see working with the capillaries in that instrument stem from pipetting accuracy. Most times the reaction mix requires you to pipette very minute volumes that increase error. Diluting the DNA sample down/changing reaction mix so that you have to pipette at least 5ul of template is a good starting point. Diluting will also dilute any impurities that may be in your eluted DNA.
I am not an expert at all in real time PCR. But I would try normal PCR, and running a gel (1,5% agarose or so) to check the quality of your DNA. ANd I will try the PCR with different dilution: 1/10 up to 1/1000. Because if you have quality DNA and no amplification it could be an inhibition problem. It happens to me for normal PCR and Real-time PCR. I had amplification problem link to inhibition with some sample and and with other. And they were extract from the same animals (mussel gill) and same kit. For some samples results became consistant with 1/1000 to 1/10 000 dilution.
And I also had problem with triplicate in splitting the reaction. You should vortex them well. Pipetting mix is not enough. I had better result doing 3 differents replicates and not splitting 1 into 3. God only knows why ;-)
And good luck! because I finally decide Luck is also important as anything else!
I have a similar problem and would like some help.
My main problem is that some of my samples don't even have melting temperatures (even in replicates). I wonder why some of the duplicates have melting temperatures and why some don't. My other problem is that even my duplicates with some very similar Tms (76.89, 76.86) turn out with different CPs (39.42, 37.41, respectively).
Then, for some samples, I get 2 melting temperatures. Even in duplicates, sometimes there's one and sometimes there are two Tms (sometimes I get it, sometimes I don't). I wonder whether the 2 Tms had an effect on such inconsistent results.
Thanks for all the help!