I have also attached the thesis.

I use this technique all the time, and it works fine. But I have modified the conditions. What is your PCR reaction set up - what reagents and what volumes do you use? If you can say that then it might be easier to help.

I have used Richard Cawthon protocol to determine the telomere length. I had the same problem at the intiail stage, when I tried to standardize my protocol. When I use Abion Turbo DNAase to clean up my RNA. It actually solved my problem. I did not find any signal in the negative control.

I use the RT PCR machine Lightcycler 480 for my experiments.

I have tried the following PCR reaction setups:

SYBR mix(Master Mix from Kappa)

Tel FP- 0.5ul(from a 10uM stock)

Tel RP - 0.5 ul

hAlb FP - 0.5ul

hAlb RP - 0.5ul

H2O - 1ul

DNA- 2ul

for DNA I start with an initial concentration of 20ng and use dilutions from 1:125 to 1:4000.

my reaction is as follows:

94C- 15sec

49C - 15sec 2 Cycles

94C- 15 sec

62C- 10 sec

74C- 15 sec

84C- 10 sec

88C- 15 sec 32 Cycles

Melting Curve

94C - 1 mins

55C - 30 sec

to 94C

to check whether I am

I have also tried the Protocol with Qiagen quantifast SYBR Green and LightCycler 480 SYBR.

I have extracted the DNA from a Column kit will it still give a RNA contamination?

I dont think so . coumn kit works better than anyother method. I have used column too and it works very well. I used the same protocol that u have mentioned. Are you trying to amplfy both reference gene and target gene in same well? if so try to amplify them in different well. Since I had limitation in my funding. I do not have resources to standardize by amplifying both genes in the same well (as richard cawton demonstrated in the paper) regrding the quantity of DNA.It depends on ur target gene. I worked in aging gene, which expression will go down as aging occur. so I di not find amplification when i used 20, 35 so I used 50ng. I hope this willhelp.

I set up 3 types of reaction every time.. one with only telomere primers, one with only alb primers, and one with both... i get a negatvie band in all...

did u check your DNA quality? If there is more salt concentration then it might inhibit the PCR amplification.What INSTRUMENT ARE U USING? I used

Bio - Rad MyiQ single color Real time PCR detection system.

Primer for telomere length and scg albumin gene (IDT Technology).

Quantifast SYBR GREEN kit (Qiagen Cat no: 204058)

Did u check ur melt curve. If your melt curve?

I had this problem too! So I followed the conditions discrebed in this link bellow and the reaction is working very well. (they tested with blood cells, but it wokrs also with my endometrial tissue samples)

http://b2b.qiagen.com/literature/qiagennews/weeklyarticle/11_04/telomere/default.aspx

Hallo Prateek Arora, i have the same problem, signal in the negative control, did you solve it?

Thank you!

Stefano i changed my project because of this problem... so sorry wont be able to help you..

you should reduce number of cycle for tel to 22 cycles and for hbg to 25cycles

I have the same problem...any solution?

There are a couple of inherent problems due to telomere (TTAGGG) special repeat sequences. However, the signals with negative control should be mainly due to primer dimerizations. Even this problem is resolved, Cawthon's Telomere Length Assay is not working for quantitative telomere length assay. There are some PROPRIETARY INFORMATION on the issue which I don't want to disclose now, but everyone may consider: Would it be same to measure (using Cawthon's q-PCR) 10 molecules of 100-bp DNA fragment vs ONE molecular of 1000-bp one given two DNA molecules contain the same (TTAGGG)n sequences? In Cawthon's protocol it seems the same but in fact, it NOT following RT-qPCR principle. Those claims "measurements of absolute telomere length using RT q-PCR" (Nathan J. O'Callaghan 2008; 2011) are totally wrong!