Inconsistent results from replicates in Taqman real-time PCR - (Jun/24/2006 )
Hi all,
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?
Help me....
Chris
Alls sugestions of other people are good,
If Ct's are late (above 30) you inherit get larger differences in replica's because of differences in effeciency + reagents get used up.
So if you can, introduce more template and it might help.
Theoreticly if you dubbel your template, your Ct drops by 1
Some mastermixes work better if you go from 1x to 0.8x (indeed lowering enzym+ buffer can increase the efficiency of your reaction). Naturally you would think opposite.
If your reaction effeciency is not good enough, then try to change your primer and/or probes concentration if you use them. Try a lower Ta les specific, but makes reaction easier.
Bottem line is optimise your reaction as best as you can,
toy around with primer conc. probe conc., different combinations, different Ta, longer elongation times. Some hotstart enzymes need 15 min to get activated, so if your initial denaturation is only 5 or 10 minutes you see your effeciency drop like hell.
lower /higher MgCl might work, I even used 4% DMSO final conc. with succes to lower Ct values (DMSO gives your ploymerase more timee and easyer acces tot the template.
If you work with genomic DNA as template (NEVER VORTEX, because this will shear your DNA and your initial copynumber drops in a non repeatable maner. With cDNA go right ahaid, vortex it.
Last but not least, and this is something I hardly ever see in forums, your own pipetting skills.
When I first started with qPCR I saw 3-4 Ct differences in replica's, if you take very good care with your pipetting (changing tips, do not insert them to deep in the solution because DNA/RNA als can be on the outside of your tip and change a lot, get low binding tips,....
Just by taking care of my pipetting skills I already got my samples withing 0.5Ct difference.
Now that I optimized my reactions (control + GOI) and combined them, I have less then 0.1 difference in Ct in true replica's. (1 sample, becomes 3 reactions).
Sorry, I tend to get my responses to grow rather large
Hope this gives some ideas
Hi All,
Thank all of you for contributing so much for this interesting topic. I'm encountering the same problem about calculating rPCR results with inconsistent results in triplicates in Taqman rPCR for samples with very low copy of target genes. The Ct are around 40. I can not increase the template amount into PCR reaction mix simply the total DNA is very limited. I split one sample into 3 triplicates and believe that my pipetting skills are good enough.
The problem now is that one well of the triplicate may show a Ct of 38, another 39, while the third one of undetectal. The worse is seen in those samples with one of triplicate with a Ct of 38, and the other 2 wells of undetectable. I'll be very much grateful if anyone could suggest how to quantify those samples, if we choose not to reclassify them as binary data, i.e., positive vs. negative.
Help me and thanks in advance!
madeinspain -
You may want to try diluting your template with NF water 1:2 or 1:4. This is an easy solution that can decrease inhibition that may be present from your sample prep. I would try dilution before pursuing more complicated steps. I have seen an decrease in cT values in samples diluted 1:4, which is counterintuitive, but by diluting out the inhibitors, the PCR runs more efficiently, resulting in lower cT's even though you have added less template. Hopefully this will give you more reliable data. . . . .
I'm affraid for the Ct values around 40 there is so much internal PCR variability there's nothing that could help you. I personaly wouldn't quantify them at all.
I'm affraid for the Ct values around 40 there is so much internal PCR variability there's nothing that could help you. I personaly wouldn't quantify them at all.
I'm affraid for the Ct values around 40 there is so much internal PCR variability there's nothing that could help you. I personaly wouldn't quantify them at all.
Thank you, Trof, Yes, we agree with you. Interestingly, Narayan Shivapurkar and coauthors could detect genes in linear relation until cycle 44 (Cancer letters, 2007; 247: 56-71). in fact, the first standard point in their standard curve construction started with cycle 36, with serial dilution, their most diluted standard point (SD6) reached Ct 44. In our lab, the Ct of the most diluted standard point never reached so high Ct, still less the huge CV in replicates for those very diluted standard points. Any tips how to make the last few standard points (I mean more diluted SD points) visible with reasonable CV in replicates?
rPCR7.
Hi everybody,
Your discussion is very interesting, and I have a similar problem. When I run QPCR for my HKG (GAPDH and Rs 15) I dont get similar curves for the values calculated with the standard curve method, and I dont get similar values for different time points. However, my C(t) values are quite stable for both genes, and for different time points. Could anyone help me with this? I did different RT, and different PCR, but always the same results.
P.S. I am using BioRad Miniopticon with SYBR Green.
Thank you so much...