non replicable Ct number and a lot of other problems

[atlantide]

Hi all!
real- time PCR is going to make me crazy! I just started with the method in january.

Here is what I do: Real-time PCR wirh Taqman probes and primer for 16S rRNA. They have allready been used succesfully.

What I do: I run my samples in triplicate. So on a plate I load my my PCRmix (with probe and primers, everythings) on the fist column (72 µL) and then add the samples (3µL). Then I mix with pipetting and divide it in the next 2 columns. So I should have (25µL) for each triplicate.

I use filtertips, multichannel pipet, work on ice and centrifuge my plate to avoid air bubbles.

I use different dilution of the same sample. And do each time a dilution series from a standard stock of E. coli 16S rRNA.

I have différents kind of problems:

First I do not have the same Ct number between my triplicate. (I have check my pipetting and it seems to be ok)


Second I have worst results now after a few try!! Now out of the triplicate I have very often only one wells with amplification! And nothing in the other. I do not understand how it is possible since I should have the same in the three triplicates.


Last, since I am starting the methods I always use the same samples. And the results make no sens: Between dilution I do not have corresponding difference between my Ct number. And between run. I also do not have the same number of copies at all.


So if anyone has an idea of what I do wrong, or what I should do... or Should not do. Thanks a lot. If you need more informations, please ask

Caro

[stefulj]

I used to have unreproducible results when having too high Ct-s (30 or higher). What are your Ct-s? Adjust the cDNA amount if this is the case (or consider cDNA/RNA degradation)....
Also, _I_ don't belive in mixing with pipetting, I prefere mild vortexing...
Hope something works for you!
stef

[atlantide]

My Ct are usually between 20 and 30 so I suppose it is fine.

And I just found out that I might have some inhibitors in my DNA so the more diluted replicates works better.

I have indeed change from pipetting to vortexing and now my results are really improving. I think I still need practice but sooner or later it will be allright.

Thanks for your advice!

Caro

[masterdiego]

hello, i think you have to review your standar curve!! may be if you change the theeshold in you standar curve you Ct is more replicable

[Silverwolf]

I have a stupid question... how do you manage to work on ice??
The instructions state we should but i found it impossible. I work on ice on all steps except from the point where i add the "master mix" to the plate. I was told not to let anything touch the wells since that would mess with my readings. Are they over-reacting?

[PeterScherp]

Hi,

first of all, I also do not believe in mixing by pipetting. I also tried vortexing which is ok. THe BEST result I got so far is this: Setup your master mix with DNA in a 1 ml centrifuge tube, mix by gently inverting the tube 3 - 4 times and spin it down real short in the centrifuge. Then load the wells...I found that that works the best, at least for me.
Also, the inhibitors may be the answer to the solution. You probably have an ethanol extraction step in you isolation protocol. If this is so, dry down your DNA sample in a Speed-Vac to complete (!!!) dryness. Then resuspend in water or buffer, whatever you want. That works best.
Also, consider that for realt-time PCR you need very little concentration of DNA and primer. Some manufactureres recommend 50 - 100 nm primer per reaction.
Too muhc primer can result in hinderance during PCR and give bad results.

[atlantide]

Thanks for your different answers.

I have triplicates which have the same values now, but still problems with the efficiency. We are working on that. And I give it a break because it is so fustrating and I am working on (not frustrating?) clone library... A colleague continue the QPCR.

masterdiego: I try playing with the Ct but it does not change anything.

silverwolf: To work on ice is quite easy for me. I have two ice basket, one with the reagent and template, and one with my plate. I keep it all the time on ice untill I load it in the cycler.

PeterSherp: I have found another solution which works fine for mixing: I first load the plate with the template (1 or 2 µL). Then do the master mix, vortex it, and load the plate. The big volume of master mix is enough to have everything mixed properly. then I shortly centrifuge my plate (30-60s).

As I use a Kit for the extraction and follow exactly the manual it should be OK... Next time I will perhaps add a washing step. Or I may try speed vac.

I will also try with less primer thanks!

Caro

[AnnaP]

HI everybody

I am haveing some problems with late Cts as well, and I have a question about what "stefulj" said about ...(or consider cDNA/RNA degradation)....
please stefulj, can you explain further??
cheers
Anna

[masterdiego]

[quote=atlantide;3079]Thanks for your different answers.

I have triplicates which have the same values now, but still problems with the efficiency. We are working on that. And I give it a break because it is so fustrating and I am working on (not frustrating?) clone library... A colleague continue the QPCR.

masterdiego:
Hello i think to eliminate the problem with the efficiency, you must to prove another set of primers and check the program used. Another important point or approach is check the denaturation curve! if your primer can form dimers with the concentration used (you can dilute the concentration and avoid the primers-dimers) and more important check if you have only one PCR product, all this factors affect the efficiency in your experiments.

[stefulj]

hi Anna
low amount of starting templete due to RNA or cDNA degradation (during preparation or storage) might be a resaon of late Cts. hope this explaines what I ment to say...
good luck
stef