I am trying to set up aTL qPCR experiment following Callaghan's paper (doi: 10.1186/1480-9222-13-3) and was wondering if anyone had bad luck with the standard curve generation?
Our set up is Power SYBR Green master mix from Thermo and QuantStudio 12K flex machine with 384-well block.
Everything seems to work fine except for the telomere standard curve from synthesised oligo ((TTAGGG)14). Ordered from Sigma with HPLC purification.
From the highest amount of 60 pg per reaction I get amplification at cycles 25-26 and the curves look really flat. No real amplification at 1:10 dilution and beyond.
Also it seems that the fluorescence initially goes up from negative to positive values at cycles 1 to 5, but then drops again into minus before it levels at 0 at cycle 15-16. This never happens in the water control wells - fluorescent values are pretty much always at 0 there.
Tried several optimisation steps (various concentrations of the dilutions, modifying primer concentrations, modifying cycling parameters) but nothing seems to help.
Any insights will be much appreciated!
Thank you
I've never used a synthesized oligo for a standard curve, but I know you can get issues if your template is too dilute, too concentrated, or is degraded. How many copies are in your dilutions? What do you see if you use the oligo for standard PCR? Sounds silly, but double check your math. And don't repeatedly freeze and thaw your standards. Most folks will make them one and aliquot out amounts to use in strip tubes.
Typically, the hardest part in making a standard curve is finding primers with acceptable efficiencies. Did you order HPLC purified primers? For this sort of assay, that will really matter.
It looks like the original protocol is very well-detailed and easy to follow. Good luck!
Katie A Burnette thank you for chiming in!
Yes, weird problem, and it seems to appear only in the telomere standard qPCR. All reagents have been prepared freshly from lyophilised stocks and I am pretty sure the math is correct:)
In this setup there is a second oligo for single copy gene (36B4) standard curve that is similarly long and is diluted to the same concentrations - we run it in parallel and qPCR works just fine. Of course, 36B4 primers have unique binding sites on the template oligo with 100% match. In contrast, telomere primers have designed mismatches plus overhangs. Also if I get it right, the reverse primer can bind in two different positions on the template.
I think this problem must have something to do with the particular master mix chemistry or machine setup rather than pipetting or concentrations.
Maybe it's just a bad batch of oligo template or primers? Try ordering them again. It does sound like something is wrong with that component and not your skills or equipment.
After some testing, it became obvious that the blame goes to Power SYBR Green master mix.
I suspect that dUTPs (used for UNG/UDG contamination removal system) somehow interfere with this particular PCR, since a SYBR master mix from another company containing normal dNTPs worked pretty well (with both batches of primers - desalted and hplc-purified; and for both desalted and hplc-purified template oligo).
Hmmm. We are also doing telomere qPCR with the same absolute standards and Master Mix and everything amplifies as expected. The concentration seems good. Are you making sure the Stds are re-suspending well enough after making dilutions?
Catherine Wellman now it is getting interesting.. i attached a typical amplification plot that i see with Thermo Power SYBR. Curves that go up are replicates of the highest concentration (60pg), the other 3 curves - 1:10 dilution. no amplification beyond this. I understand that proper mixing during serial dilutions is one of the obvious things to look into, but it is definitely not the case here (i will upload some working standard curve plots when i get back to the lab tomorrow). We tried two different batches of the master mix with similar results. And I also switched to DNA lo-bind eppendorfs for this work. I also need to add that the Power SYBR master mix DOES amplify telomeres from genomic DNA samples (at Ct of approx. 22-23) - it is only oligo template that gives problems.
I realize now that we might have some copy number differences due to species. We are working on bovine DNA, whose telomeres can be around 20kb. I also misread, we use PowerUp SYBR mix from Thermo. Maybe try that.
We use, from the same paper, the same ((TTAGGG)x14) for our absolute standard. Have you tried increased concentrations for the std curve to see if that did anything? Otherwise, I'm thinking it's the thermo cycler protocol, the primers or the thermo cycler itself. Our telomere primers are from Cawthon 2009 and our thermo cycler is Bio-Rad
Catherine Wellman PowerUP SYBR mix is Thermo's updated version of what we use. It might behave differently, of course, I haven't got my hands on it yet.
The primers from Cawthon 2009 that you use are also different from Callaghan 2011 - they are designed in a way that the reverse primer cannot actually extend the template itself - it can only start the elongation when bound to the extension product of the forward primer in certain configuration. Good to know this primer pair also works on the oligo standard.
I do not know if 20 kb telomere length is a lot or not in bovine, but we have control cells (human) at around 60 kb. Falls right in the middle of the standard curve:)
I attached the standard curve example that I get with Bio-Rad Sybr master mix. I currently have this issue reported to thermo - will update if they come up with a plausible explanation.
Di Sh will be interested to know any details or tips you can give for following Callaghan's protocl.. about to start project on this,
Zaineb Akram Just avoid using the Power SYBR master mix - it does not seem to work. Thermo reckoned it was a batch-specific problem, but none of the batches I tried worked for the telomere standard curve. On the other hand, PowerUP master mix works very well (as well as the SYBR master mix from Bio-Rad). I also found that omitting plasmid DNA from standard curve reactions does not have any real influence on the standard curve precision. Apart from this, the only thing I can recommend - use a lot of replicates (biological and technical). This method tends to produce rather large standard deviations for the same samples run on different plates/days.
Di Sh thank you so much for valuable suggestion . i will definitely keep it in consideration.
Di Sh can you guide me if we can use any of the SYBR green mix from Thermo Fisher for this particular project?
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