I am using a Real-time PCR method to evaluate the telomere length in mice tail samples (Unknown Samples). I have used the following publication as a source of the protocol.

Article A quantitative PCR method for measuring absolute telomere length

I will point out some of the required information:

• Each reaction has a final volume of 20 ul containing 1uM F+R primers per reaction, 20 ng DNA per reaction, 10 ul 5XMasterMix, and some water.
• I have the expression of telomere and 36B4 in my positive controls and my standard samples
• Most of my Unknown Samples neither express 36B4 nor telomere.
• 36b4 Single Copy Gene (200 pg to 0.002pg) or 60 pg to 0.0006 pg Telomere Standatrd per reaction almost all have expressions that indicate the primers are working well. however, the telomere primer rarely has given us perfect results.
• Most of my Unknown Samples show a concentration between 10 ng/ul to 70 ng/ul.
• Most of my Unknown Samples are showing the A260/A280 ratios of around 2 or a little less or more.
• The positive controls are having the A260/A280 ratios of ~1.86
• Unknown Samples of DNA were solved with AE buffer and further diluted with DW.
• Samples were kept in a -80 or 4-degree refrigerator for a week before performing the experiments.
• In a reaction, an Unknow Sample was loaded with 2uM primer per reaction but there was no difference.
• An Unknow Sample was diluted with AE buffer instead of DW and there was no difference.
• Some of our reagents have passed their expiration date.
• I extracted the DNA with the Qiagen DNA extraction kit.

Considering all these factors, do you think the problem has been with the DNA extraction step?

I assume there should be RNA contamination in my samples as I can see the A260/280 ratio of 2 in most of my Unknown Samples, even the Unknown Samples which are expressing.

Please comment and help me out with troubleshooting.

#PCR

#Real-time

#telomere

#telomerelength

#36B4

#primer

#RNA

#contamination

#mice

#BALB/C