The overall goal of this procedure is to provide a short, accurate, and cheap method to assess telomere length and telomeres activity in multiple tissues and species. This is accomplished by first isolating genomic DNA for telomere length determination using quantitative real-time PCR. The second step is to isolate and ly cells of the same tissue in a buffer that preserves telomerase function and to quantitatively measure telomerase using real-time PCR.
Ultimately, both telomere length determination and telomerase activity detection are used to find the relationships between telomere length and cell senescence possible mechanisms of telomere shortening and recovery responses to aging and states of cell proliferation. I was first introduced to this methods when I was looking for a fast and easy way to measure ome lengths and omera activity. The implication of this technique extender toward diagnosis of age associated diseases and premature death because its report on cell status, which reflects on organ condition and overall body shape.
Demonstrating of this procedure will be Mr.Ur Bogo, a technician of my lab. To begin this procedure, isolate DNA from the subject's blood using the Qiagen DNE kit, specifically formulated for blood sources at the final step. Elute the DNA in PCR grade water and measure the concentration using a spectrophotometer.
Next, prepare stock PCR primers as described by OC Callahan et al for the single copy gene or SCG control and for the telomeres by diluting them to 100 picomoles per microliter. Using PCR grade water while the primers rehydrate, prepare serial dilutions of the isolated DNA. Begin by diluting the stock DNA with PCR grade water to a top concentration of 6.1 nanograms per microliter for telomere reactions and 1.8 nanograms per microliter.
For SCG reactions, let the dilution sit for a minimum of 15 minutes before moving on to the next one. After 15 minutes, prepare the next dilution according to the table shown here. Repeat this for each dilution listed.
Next, set up the PCR reactions in a standard PCR plate. Each sample, along with the SCG controls are run in triplicate. First, add six microliters of PCR grade water to each well then add 10 microliters of the cyber green reaction mixture.
Then add the primers in the amounts shown here to their respective wells. Next, further dilute an aliquot of each of the stock primers with PCR grade water tenfold so that the final concentration is 10 picomoles per microliter. Finally, add two microliters of the serially diluted DNA to their respective wells and cover the plate with an adhesive plastic sheet to seal in the contents.
Then place the plate into the real-time PCR machine. Next, prepare a program on the Roche four 80 light cycler, beginning with a 10 minute denaturation. Step at 95 degrees Celsius, then cycle 95 degrees Celsius for 10 seconds, 60 degrees Celsius for five seconds and 72 degrees Celsius for 11 seconds.
For a total of 50 cycles, perform cyber green measurements after each cycle. Next, begin the program once the thermocycler has completed. Set the CT line at the beginning of the exponential curve and record the cycle values from when each sample crosses the threshold line.
Next, calculate the ratio of cycle numbers between the telomere and single copy gene samples. Since the telomere has multiple regions, which will amplify in each cycle, telomere CT values will be smaller than the single gene copy number resulting in a ration of less than one. Finally, calculate the telomere length based on work by Terry Etal, where one telomere per single gene copy ration unit is equivalent to a mean telomere length of 4, 270.
Base pairs in leukocytes perform telomerase activity measurements using the quantitative telomerase detection from allied biotech to begin measurements, collect 10 to the fifth to 10 to the sixth peripheral blood cells and rinse them in PBS. Then pellet them at 500 times G for 10 minutes at room temperature. Next, resuspend the pellet in 200 microliters of one x lysis buffer from the assay kit and incubate on ice for 30 minutes.
Following incubation, centrifuge the samples at 12, 000 times G for 30 minutes at four degrees Celsius. To pellet the cell debris transfer 160 microliters of the resulting supernatant into a fresh tube and flash, freeze the remaining supernatant in an ethanol and dry ice bath before storing it at minus 80 degrees Celsius for repeat use. Next, determine the protein concentration of the supernatant by standard BCA assay.
For each sample collected, calculate the amount of protein needed for the assay based on the working range of the telomerase kit. Then since telomerase is a heat sensitive enzyme, heat inactivate half of each sample for use as the negative control by incubation at 85 degrees Celsius for 10 minutes. Next, prepare the quantitative standard curve by serially diluting stock.
TSR Oligonucleotides dilute the stock solution five times at a one to five dilution using lysis buffer. TSR Oligonucleotides have an identical sequence to telomere primers and are of a known concentration. Next, set up the master mix consisting of 12.5 microliters of two x quantitative telomerase detection premix, and 11.5 microliters of PCR grade water per sample and mix.
Then pipette 24 microliters of the master mix into each tube being used. Once the master mix has been dispensed, add one microliter of each template consisting of either the standard, a heat and activated sample or a regular sample to the wells. Next, seal the plate with an adhesive plastic sheet and mix the samples by vortexing.
Then place them into the real-time PCR detection system. Set up the PCR program to begin at 25 degrees Celsius for 20 minutes. During this time, the telomerase reaction will take place.
Follow this with 95 degrees Celsius for 10 minutes for the initial PCR activation step. Then perform 40 cycles of 95 degrees Celsius for 30 seconds, 60 degrees Celsius for 30 seconds and 72 degrees Celsius for 30 seconds. To amplify the telomere templates added during the 20 minutes at 25 degrees Celsius.
Finally run the program and collect the threshold cycle values for each sample. Then generate a standard curve using the CT values and use it to calculate the telomerase activity. In the samples shown.
Here are realtime PCR results of a 65-year-old subject. The telomere gene represented by the bright red lines reaches its exponential curve after about 27 cycles. The second set of lines represents the single copy gene amplification, which has only one copy in the genome, and thus has fewer copies than the telomere samples represented by the red lines.
Also, the single copy gene reaches its exponential curve after about 31 cycles. The brown lines represent the 33 standard curve based on a serial dilution of known sample concentrations. The standard curve for the log of concentration versus threshold values is shown here.
A straight line confirms that the serial dilution was accurately measured and loaded. The results of the real-time PCR analysis is then used to estimate telomere length. One telomere to single gene copy ratio unit is equivalent to a mean telomere length of 4, 270 base pairs in leukocytes and from this data results in a ratio of 0.87, which is an average of 3, 715 base pairs and intermediate length for someone of this age.
Using results from the quantitative real-time PCR assays, a relationship between telomeres activity and the telomere to single gene copy ratio can be elucidated. This graph shows a strong relationship between the two assays After its development. This technique paved the weight for researcher in the aging field to explore cell senescence in modal organism, patient demographics and organ systems.
