40 replies to this topic

[Tom-S]

[quote name='dtae' timestamp='1290711988' post='93168']
I'm not sure what's up, but hear are a few thoughts:
1. Some people are publishing with low efficiencies and using LinRegPCR to correct for this. The greater than 90% efficiency rule of thumb seems to be just that, a rule of thumb with limited empirical basis. The fact that the E between T and S amplicons are similar is reassuring.
2. The sample you are using to make your standard curve might have inhbitors/enhancers in it. Try to exclude low or high C samples and see what it does to efficiencies. You might also try using different samples to construct your standard curve with.
3. See what kind of well-specific efficiencies you are getting. You might find that those are substantially different than using the standard curve method.
4. Beware of well-position effects--they can cause unpredictable and unstable efficiencies.
5. Ct CVs really don't tell you a great deal. T/S ratio CV is what really matters here.


Many thanks Dan,

These experiment are very frustating for me as i'm convinced that i'm doing things quite well...! Your remark concerning the use of different DNA to construct standard curve. Concerning the point 4, each time I carefully checked that my samples are always at the same position in pairs for T and S.
I will work on suggestions and keep you (and the others investigators of qPCR for telomeres) inform.
Have a nice week-end

[dtae]

see here for more info on what i mean about well-position effects:
http://www.protocol-...ffects-in-qpcr/

[shvir]

I have tried Cawthon's multiplex qpcr method with his newest albumin primers and thermal cycling profile and have found that the albumin gene amplification overlaps with the telomere amplification instead of rising above threshold at a later cycle number than the telomere. In his 2009 paper, the purpose of the multiplex was to thermally separate the two amplifications in the same reaction well, but with these newest primers, albumin amplification occurs at the same cycle number as the telomere and crosses the threshold at the same time as well. Has anyone else used the newest albumin primers posted on this forum and had this problem at all?

I appreciate any response/help. Thanks in advance.

Edited by shvir, 14 January 2011 - 01:44 PM.

[dtae]

I forget what the problem was, but I got better results with the old albumin primers than the new ones. Are the two products distinct on the melt-curve?

shvir, on 14 January 2011 - 01:34 PM, said:

I have tried Cawthon's multiplex qpcr method with his newest albumin primers and thermal cycling profile and have found that the albumin gene amplification overlaps with the telomere amplification instead of rising above threshold at a later cycle number than the telomere. In his 2009 paper, the purpose of the multiplex was to thermally separate the two amplifications in the same reaction well, but with these newest primers, albumin amplification occurs at the same cycle number as the telomere and crosses the threshold at the same time as well. Has anyone else used the newest albumin primers posted on this forum and had this problem at all?

I appreciate any response/help. Thanks in advance.

[shvir]

The products are distinct, however the telomere amplicon melting peak is lower than it should be for samples. In a previous post where Cawthon's direct response was quoted, he explained how this occurs due to the GC-rich scg product stealing the SYBR Green.

Just to clarify, which "old" primers worked the best for you? The ones published in his paper or the first "new" set of albumin primers previously posted in this forum (albugcr1, albdgcr1)? The "newest" primers I'm referring to with this problem were labeled albugcr2, albdgcr2.

dtae, on 14 January 2011 - 01:50 PM, said:

I forget what the problem was, but I got better results with the old albumin primers than the new ones. Are the two products distinct on the melt-curve?

shvir, on 14 January 2011 - 01:34 PM, said:

I have tried Cawthon's multiplex qpcr method with his newest albumin primers and thermal cycling profile and have found that the albumin gene amplification overlaps with the telomere amplification instead of rising above threshold at a later cycle number than the telomere. In his 2009 paper, the purpose of the multiplex was to thermally separate the two amplifications in the same reaction well, but with these newest primers, albumin amplification occurs at the same cycle number as the telomere and crosses the threshold at the same time as well. Has anyone else used the newest albumin primers posted on this forum and had this problem at all?

I appreciate any response/help. Thanks in advance.

[dtae]

I've had best luck with the albumin primers from the originally published paper. I forget right now if I tried both of the others, or just the newest one. Avoid the beta-globin primers except for cross-validation (they have a mess of SNPs in them and I think are found in multiple copies across the genome).

d

shvir, on 14 January 2011 - 01:56 PM, said:

The products are distinct, however the telomere amplicon melting peak is lower than it should be for samples. In a previous post where Cawthon's direct response was quoted, he explained how this occurs due to the GC-rich scg product stealing the SYBR Green.

Just to clarify, which "old" primers worked the best for you? The ones published in his paper or the first "new" set of albumin primers previously posted in this forum (albugcr1, albdgcr1)? The "newest" primers I'm referring to with this problem were labeled albugcr2, albdgcr2.

dtae, on 14 January 2011 - 01:50 PM, said:

I forget what the problem was, but I got better results with the old albumin primers than the new ones. Are the two products distinct on the melt-curve?

shvir, on 14 January 2011 - 01:34 PM, said:

I have tried Cawthon's multiplex qpcr method with his newest albumin primers and thermal cycling profile and have found that the albumin gene amplification overlaps with the telomere amplification instead of rising above threshold at a later cycle number than the telomere. In his 2009 paper, the purpose of the multiplex was to thermally separate the two amplifications in the same reaction well, but with these newest primers, albumin amplification occurs at the same cycle number as the telomere and crosses the threshold at the same time as well. Has anyone else used the newest albumin primers posted on this forum and had this problem at all?

I appreciate any response/help. Thanks in advance.

[shvir]

Has anyone validated the Multiplex by comparing Ct values to the Singleplex Ct values for each primer pair?

[dtae]

I believe Cawthon did in his paper..and I've found a nice strong correlation as well.

shvir, on 24 January 2011 - 01:57 PM, said:

Has anyone validated the Multiplex by comparing Ct values to the Singleplex Ct values for each primer pair?

[shvir]

I meant independently of Cawthon, just to make sure. Did you have to supplement your multiplex with anything or did you use the exact same concentrations/conditions for the singleplex as the multiplex and get a good correlation? I'm not sure why I'm not getting a great correlation so I wanted to hear if others did anything differently.

Thanks!

[dtae]

How do your standard curves and CVs look using both multiplex and singleplex versions? Are your melt curves good? Is there several cycles seperating the Cq values in T and S in multiplex?

Dan

shvir, on 24 January 2011 - 02:40 PM, said:

I meant independently of Cawthon, just to make sure. Did you have to supplement your multiplex with anything or did you use the exact same concentrations/conditions for the singleplex as the multiplex and get a good correlation? I'm not sure why I'm not getting a great correlation so I wanted to hear if others did anything differently.

Thanks!

[shvir]

Everything is great, melt curves, cycles between T and S in multiplex, standard curves, efficiencies. The only thing that's not happening correctly is my multiplex is off from the Ct values of singleplex in varying amounts, from a 0.2 difference for one sample to 1.3 for another! This variability is consistent. I'm thinking it might be that I'm not careful enough with inputting DNA maybe (although I use a multichannel pipetter) or that I need to make more master mixes for a smaller number of samples, but if you have any other suggestions, I would be glad to hear it.

Thanks for your responses!  I really appreciate them!

[dtae]

A few ideas:
- be careful of well position effects - I think I covered this in other areas of this thread or in other submissions to this message board.
- How do your T/S ratios look comparing between multiplex and singleplex? That is more important than Ct values directly
- What are you CVs like across replicates?

D

[shvir]

Great ideas! The assay works fairly well now. I'm wondering what you concluded about efficiencies with everyone's input? My efficiencies aren't bad, but I'm wondering if I should re-run samples if my telomere efficiency is 87% and my albumin is 102% (I accept 105% as my upper limit)

[dtae]

The most important thing with efficiences is probably that your control sample(s) and unknown samples have similar efficiencies in both telomere and scg amplicons. As long as you are correcting for efficiences in your calculation method, I don't think having different efficiencies between telomere and scg is a big deal.
I would suggest looking at your efficiencies on a well-by-well basis using LinRegPCR as well as the standard curve method. I bet you'll get substantially different results.

Dan
Department of Anthropology
Northwestern University
www.dtae.net

[shvir]

I've looked through so many papers using this protocol and am getting confused on what the correct method is to calculate T/S ratios.


In his original paper, Cawthon stated, "T/S ratio is approximately [2^Ct(telomeres)/2^Ct(36B4)]^–1 = 2^–ΔCt" and that the relative T/S ratio is 2^–ΔΔCt. "Using this formula, a relative T/S ratio for each of the 96 wells was determined, as the T/S of the well relative to the mean T/S for all 96 wells."

In his multiplex paper, he states "... was used to generate two standard curves for each plate, one for the telomere signal and one for the scg signal. The T/S ratio for an experimental DNA sample is T, the number of nanograms of the Standard DNA that matches the experimental sample for copy number of the telomere template, divided by S, the number of nanograms of the standard DNA that matches the experimental sample for copy number of the scg." which means just using the standard curve to determine the amount of DNA in a sample.

In other papers by other authors that have used this multiplex protocol, the T/S ratio was divided by a calibrator sample (for example, DNA from a cell line known to have short telomeres).

I'm wondering if all these methods are interchangeable. I've tried these analyses, however, and get different ratios. So my question is, what is the most appropriate way to calculate these T/S ratios?