Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

The overall goal of this procedure is to measure telomerase activity in normal brain tissue. This is accomplished by first preparing whole cell protein extracts from various brain tissues as the source for an active telomerase enzyme. The second step is to add equivalent amounts of the protein extracts to a specific telomerase reaction mixture containing the substrate and nucleotides needed for the telomerase activity.

Next, the telomerase reaction products are amplified by PCR and analyzed by high resolution aros gel electrophoresis. The final step is to visualize the amplified telomerase reaction products by staining the aros gel with a highly sensitive DNA staining agent ultimately results from this thoma ray's repeat amplification protocol assay show six base pair increment, DNA ladders equivalent to thoma ray's activity in various brain tissues. The main advantage of this technique over existing methods, like a radioactive trap assay or commercial trap assay kits, is that this protocol offers a relatively time and cost efficient alternative.

Though this method can provide insight into thous activity in brain tissue, it can also be applied to any other living tissue or cell culture. Prior to starting this procedure, prepare three 15 milliliter tubes each containing five milliliters of ringer solution. Place the tubes on ice after removing the brain from a euthanized mouse.

Rinse the brain for a few seconds with ice cold ringer solution to prevent damage to the brain tissue. Using a surgical blade, separate the cerebellum and brainstem from the frontal cortex. Next, separate the cerebellum from the brainstem.

Remove the olfactory bulb from the frontal lobe and cut the frontal lobe from the frontal cortex. Place the three brain regions in the designated 15 milliliter tubes, homogenize the tissue in each tube. Next, add fresh ice cold ringer solution so that each tube contains the same volume and centrifuge for seven minutes at 800 times G, remove the supernatant and add 100 microliters of chap slices.

Buffer to each tube. Mix well by passing the solution about 10 times through a one milliliter syringe with a 21 gauge noodle incubate on ice for 30 minutes. After 30 minutes, transfer the contents of each tube to a new micro centrifuge tube and centrifuge for 30 minutes.

At 13, 000 times, G, transfer the supinate to a new micro centrifuge tube. After determining the protein concentration using a previously established method store the protein extracts in aliquots of 10 microliters at minus 80 degrees Celsius. Begin this part of the protocol by preparing the appropriate number of labeled micro centrifuge tubes for the protein extract dilutions and the telomerase reaction buffer for the following solutions on ice.

10 x trap reaction mix DS primer dn tps, ultrapure water and protein extracts dilute the solution of the protein extract to a final concentration of one microgram per microliter. Using ultrapure water perform all pipetting using filter tips to prevent contamination or the telomere reaction. Each sample requires a telomere reaction mix that consists of two microliters of trap Mix one microliter of TS primer, one microliter of DN NTPs, and 15 microliters of ultrapure water for multiple samples.

Multiply these volumes by the number of samples plus one, add 19 microliters of the MASE reaction mix to each reaction tube, followed by one microliter of the diluted protein extract. For a final volume of 20 microliters, place the reaction tubes in a 30 degrees Celsius preheated water bath for 45 minutes. 15 minutes before the end of the thres reaction, prepare for the amplification of the thres reaction products by hawing the following solutions, a CX primer, titanium tack buffer, and ultrapure water.

Each sample requires a PCR reaction mix consisting of 2.5 microliters of titanium tech buffer, one microliter of a CX primer, two microliters of ultrapure water and 0.5 microliters of titanium tech polymerase for multiple samples. Multiply these volumes by the number of samples plus two. Add six microliters of the PCR reaction mix to each PCR tube and add the entire volume with the mase reaction to each tube.

For a final volume of 26 microliters, insert the PCR tubes into a PCR thermocycler and run the following program, 90 degrees Celsius for two minutes, followed by 34 cycles of 94 degrees Celsius for 30 seconds. 60 degrees Celsius for 30 seconds and 72 degrees Celsius for 45 seconds. And a final two minutes at 72 degrees Celsius.

When the PCR reaction is complete, store the PCR products at minus 20 degrees Celsius. To prepare the egg roses for casting the mini gel first, add 25 milliliters of chilled TBE Electrophoresis buffer and a stir bar to a beaker that is two to four times the volume of the buffer solution and start stirring. Then slowly sprinkle in 1.125 grams of the high resolution aros powder.

While the solution is rapidly stirring, remove the star bar. Cover the beaker with plastic wrap and pierce a small hole for ventilation. When hitting the agros, it is critical to reach the boiling point to dissolve the agros completely.

Be sure to give full attention to the beaker in the microwave to make sure the foam generated won't spill over. Heat the beaker in the microwave on low power for three minutes after that switch to high power and heat the solution in short pulses using care to avoid spilling. Cast the gel in a horizontal mini gel apparatus and let it solidify at room temperature After the gel has solidified gel for 20 minutes at four degrees Celsius.

Before use for electrophoresis, load each lane with 25 microliters of trap sample and run at 110 volts for three hours in a four degrees Celsius cold room while the gel is running. Prepare a three x nucleic acid stain working solution by adding 15 microliters of 10, 000 x nucleic acid stain stock solution to 50 milliliters of double distilled water with 0.1 molar sodium chloride. When the electrophoresis of the samples is complete, stain the gel for 20 minutes at room temperature with gentle shaking.

Lastly, use a UV trans illuminator with a 302 nanometer wavelength. To view the trap ladder, DNA bands telomeres activity was detected in regions of the mouse brain by both the traditional and modified trap assays. Better visualization and resolution of the telomeres DNA products was observed with the modified assay as seen by the length and intensity of the DNA ladder.

A gradual reduction in the telomere DNA products was observed when decreasing concentrations of protein extracts were used in the modified trap assay suggesting its sensitivity for the detection of telomeres activity at lower concentrations of proteins, detection of telomerase activity by the modified assay in three brain regions of one month old female and male mice showed no significant differences. However, in three month old mice, higher telomerase activity is shown in the frontal lobe and brainstem compared to the cerebellum as revealed by the intensity of the DNA bands and the length of the DNA products ladder. During the mouse transition into adulthood, thoma ray's activity increases in the frontal lobe and decreases in the brainstem and the cerebellum.

Ome telomere activity also appears to be higher in the frontal lobe in females. In contrast to higher telomere activity in the brainstem and cerebellum in males Once mastered, this technique can be done in six to seven hours if it is performed properly. After watching this video, you should have a good understanding of how to easily determine thoma activity in your tissue of choice.