qPCR for telomere length measurement - efficiency issues - complicated monochrome multiplex assay with SYBR Green (Aug/17/2010 )

Hi, I am so glad to find this forum. I am trying to set up this Multiplex qPCR technique in the lab. I am working now. I only have access to Roche's light cycler 3. Does anyone use it before? And can any one give me suggestion on what SYBR green master mix will work best for this? If the homemade brew(Cawthon's 2009 paper) works best, can you let me know where did you order the SYBR green ? (If you can let me know the vendor and order item #, I would really appreciate!)

Thanks again. I learned a lot from this forum.

Roche's light cycler 3? I thought only Roche's light cycler 2 available?
Try cyto9 (meltdoctor, invitrogen), evagreen mastermix?

Sorry! I have access to the Roche Lightcyler 2.0, the software versio is 3.5.
The master mix I am planning to try out based on the paper, and the ordering information will be:
including( 0.75 X SYBR Green I,
<SYBR® Green I nucleic acid gel stain - 10,000X concentrate in DMSO, 500 µL, Catalog #: S7563; Invitrogen>
10 mM Tris-HCL, pH 8.3
50mM KCL,
3mM MgCL 2
0.2 mM each dNTPs DNTP10-1KT, Deoxynucleotide Set, 10 mM>

1mM DTT < Sigma; 43816-10ML; DL-Dithiothreitol solution

BioUltra, for molecular biology, ~1 M in H₂O>



1M betaine in deionized dH2O>

0.625 U AmpliTaq Gold DNA polymerase
<AmpliTaq Gold® DNA Polymerase, 250 units with Gold Buffer and MgCl2 solution; Catalog #: 4311806; or
AmpliTaq Gold® 360 DNA Polymerase, 250U;Catalog #: 4398823>


I am concering about the SYBR green I product. Can anyone let me know that's what they use? If not, what do you use?
And also about the betaine conc., the paper said 1M, is it right?

Since I will be using Lightcycler, I will try the program as Cawthron's suggested for the LC480
1. 95 degrees x 15 minutes
-----------------------------------

2. 94 degrees x 15 seconds

3. 49 degrees x 60 seconds
Repeat steps 2 and 3, one more time
-----------------------------------

4. 94 degrees x 15 seconds

5. 59 degrees x 30 seconds
Repeat steps 4 and 5, 3 more times
----------------------------------
6. 85 degrees x 15 seconds
7. 59 degrees x 30 seconds, with signal acquisition
Repeat steps 6 and 7, 19 more times
----------------------------------
8. 94 degrees x 15 seconds
9. 84 degrees x 10 seconds
10. 85 degrees x 15 seconds, with signal acquisition
Repeat steps 8, 9, 10, 26 more times
----------------------------------
Melt program: 59 - 95 degrees; 0.5 degrees, 5 seconds, per step


I already have the telg and tel c, with the original albu and albd. I might just try them out first with
Telg and telc: final conc. = 700nM each
albu and albd : final conc. = 200nM each

If not work well, I will then try
tel g: 200 nM
tel c: 700 nM
albugcr2: 700 nM
albdgcr2: 500 nM

Any suggestion?

Thank you!

I have read this forum carefully and thank you all for the great suggestions. However, now that I'm trying to reproduce Cawthon's MMQPCR I'm far from getting the desired outcome, if any outcome at all.
I'm working with the original telg/telc and albu/albd primers from his 2009 article and haven't tried qPCR yet. I'm testing the primers now singleplex with ordinary PCR to see whether I get the expected 79 and 98 bp products. When putting the PCR products on 3% agarose gel, I get this ~65 bp product in my albumin, which I'm afraid is a primer dimer of the GC clamps (estimated length 68 bp), since it's usually also present in my NTC.

For the telomeres I'm not getting reproducible outcomes. Both experiments attached were exactly the same, just performed on different days. I tried a 3 temperature PCR just in case raising the temperature to 84 and 88 degrees might interfere somehow with the reaction. (This PCR programme is similar to Cawthon's PCR, but with only 94, 62 and 74 degrees in the cycles), and compared this with Cawthon's PCR programme. Sometimes I get my telomeres only in the 3 temperature reaction, and the next time only in Cawthon's 5 temperature programme. In both attached experiments I used the standard Amplitaq Gold PCR buffer. When I tried Cawthon's recipe, I got only primer dimers and no 79 bp (or 98 bp) product at all. Using Cawthon's recipe, I tried 900, 700 and 200 nM of the primers but that didn't help.
(My DTT seemed to have aggregated because there were white pieces floating in it, but still, that shouldn't prevent the whole reaction to occur, right?)

I'm not so sure about going to the qPCR with these outcomes, because SYBR green will mainly detect primer dimers this way. Does any of you recognize these problems and what can I do about it?

-Anouk

<sub>Hi everybody,

I also try to establish Cawthon's telomere PCR in our lab. I follow the protocol given in the NAR-article from 2009.
I chose beta-globin als SCG and achieved good results concerning efficiency and R² of the standard curve.

The telomere primers (tel g/c) used to work properly as well, but right now we have problems to reproduce our results - having an average efficiency of 110-120% concerning the telomeres. I've already changed every single component of the mastermix to eliminate the risk of a possible contamination and tried to vary the concentration of the primers.
I got in touch with a technician from Bio-Rad as well, restored our calibration data and arranged a performance check of our MyiQ2. However, the cycler passed the test without any noticeable problems.

Now I don't have any other idea where to locate the source of error.
Did anyone else have similar problems with MMQPCR?

I would be glad to get some new thoughts!

Thank you!

Katrin</sub>

Primers can go bad over time. Try ordering new one.

Trof on Mon Nov 12 22:22:26 2012 said:

I already ordered new primers twice. The first telomere primers I ordered had to be resolved in only 700-800 µl aqua (synthesis scale 1.0 µmol) while the primers I used before (and which worked well!) had to be diluted in approximately 1800 µl aqua (synthesis scale 1.0 µmol as well). Consequently I mistrusted the quality of these new primers and reordered the telomere primers.

I tried both - unfortunately without results.

Hi.
I was just wondering if anyone knows which are the best primers and primer concentrations to use for this protocol. There are a number of sets that have been recommended by Cawthon, but has anyone tried all of them out?
I currently am using the ones posted in the original paper (2009) and have not been getting consistent results....
Please help me, as I have been working on this for quite some time now and am totally confused...

katrin w on Fri Nov 9 15:07:33 2012 said:

Dear Katrin,

While my qpcr used to work well , I suddenly encounter the same problems as yours. I didn't manage to find the source of the problem. I am confused now... Could you please tell me if you have found a solution?

Thank you

Hi,
I am trying as well to set up the Cawthon protocol 2009,
What i found is no signal for telomere when i perform MMQPCR. i followed the indications from R.Cawthon but i didnt use BioRad; and my power SYBR green mix is from ABI.(there is no Betaine,)i have used instead DMSO.
I run a standard PCR using the conditions from Cawthon , and i havent seem the exppected band(79bp) in the gel.
I have no problems with Albumin, and i can see the exppected band in agorose gel after running a PCR for the Albumin(98bp).
I would really appreciatte if somebody have an idea of what i am doing wrong, some recommendation?
Thanks, this is the first time i find so usefull information in a forum....Great!!!
Congratulations,