The scope of our research involves measuring telomere length differences across various study groups. The questions we are trying to answer revolve around diseases of physiological conditions and how they may affect telomere length and vice versa. The technologies currently used to advance research in the field of telomere length biology and nanopo sequencing, and more recently, CRISPR-CAS based measurement.
Current experimental challenges are accurately measuring absolute telomere length and the changes in it. The same sample could give varying results when different methods of measuring telomere length are used. The research gap we address is the lack of a standard method to measure telomere length.
Telomere restriction fragment analysis, or TRF, is still the standard against which all new techniques are evaluated. Also, our optimized protocols helps in processing a higher number of samples with limited resources. Our findings will help advance research in the field of telomere length biology by providing a reliable and relatively economical way to compare newer methods to the gold standard, which is TRF.
Our laboratory will focus on the effects of small molecule inhibitors on telomere length. We will also study the impact that lifestyle diseases and telomere length have on each other. Begin by adding 500 microliters of the lysis buffer to the previously obtained A 2780 cell pellet, and mix gently using a cut tip with a minimum opening diameter of two millimeters.
Add 20 micrograms per milliliter of freshly prepared RNAse-A and mix by gentle inversion. Incubate it at 37 degrees Celsius for 30 minutes. At the end of incubation, add proteinase K to a final concentration of 100 micrograms per milliliter and gently invert 10 times.
Incubate again at 55 degrees Celsius for one hour, inverting the tube every 10 minutes. Pipette 500 microliters of funnel chloroform isoamyl alcohol reagent into the tube and gently invert the tube about 20 times. Centrifuge it at 9, 391 G for 15 minutes at room temperature.
Pipette the upper aqueous viscous layer into a new tube. Add an equal volume of chloroform and mix by gentle inversion. After centrifuging the tube once, collect the aqueous layer.
Add an appropriate amount of five molar sodium chloride in the tube to increase the concentration by 0.2 moles. Add two volumes of 100%ethanol into the tube and invert it gently 20 times. Centrifuge at 15, 871 G for five minutes at room temperature.
After discarding the supine agent, add 500 microliters of 70%ethanol. Centrifuge again at the same condition, and then discard the supinatant. Once the pellet is air dried, add 50 microliters of sterile nuclease free water and let it rehydrate.
Then mix the DNA by pipetting using the cut tip. Use UV spectral photometry to measure the concentration of DNA. After digesting the DNA, separate it using 0.8%agros gel.
Submerge the agros gel in 0.25 molar hydrochloric acid solution for 10 minutes at room temperature with gentle agitation. Then rinse the gel twice with distilled water. To denature the DNA, submerge the gel in a solution of sodium hydroxide and sodium chloride.
Then, rinse the gel twice with distilled water. To neutralize the DNA as demonstrated, submerge the gel in a solution of 0.5 molar hydrochloric acid and three molar sodium chloride at PH seven. The extracted genomic DNA showed good integrity indicating its use for further downstream processing of telomere restriction fragments.
To perform southern blotting, start by cutting a nylon membrane into a 10 by 12 centimeter sized segment. Make a notch in the same position as the gel. Activate the membrane by dipping it in distilled water, followed by 20 x sodium saline citrate buffer.
Place a filter paper wick on a double inverted tray. Set such that the ends touch the base of the outer tray. Place the gel over the filter paper.
Then place the activated nylon membrane over the gel, ensuring that the notches overlap. Remove any air bubbles with a glass rod. Place a two centimeter heap of 9.5 by 11.5 centimeter sized cellulose filter paper and another six centimeter heap of regular filter paper.
Place a weight atop the setup for equal weight distribution. Fill the outer tray with 20x sodium saline citrate buffer, and leave it overnight for efficient transfer. After southern transfer, fix the transferred DNA on the membrane by UV cross-linking on a UV trance illuminator.
Wash the membrane twice with 25 milliliters of two x sodium saline citrate buffer. To perform hybridization, incubate the membrane in 10 milliliters of the prewarm pre-hybridization buffer. Gently agitate the membrane manually at frequent intervals.
Then, incubate the blot in 10 milliliters of hybridization buffer at 42 degrees Celsius for three hours with gentle agitation. Wash the blot twice in 25 milliliters of stringent buffer for 10 minutes at room temperature with gentle agitation. Now wash the blot twice in 25 milliliters of prewarm stringent buffer at 50 degrees Celsius for 15 minutes with gentle agitation.
Rinse with 15 milliliters of wash buffer for five minutes with gentle agitation at room temperature. Incubate the membrane in 10 milliliters of freshly prepared blocking solution for 30 minutes at room temperature with gentle agitation. Then place it in 10 milliliters of anti dioxygenen conjugated with an alkaline phosphatase working solution.
Wash the blot twice in the wash buffer at room temperature for 15 minutes. Now incubate it in 10 milliliters of detection buffer for five minutes at room temperature. After removing the excess buffer, placed the membrane on an acetate sheet with the DNA side facing up.
Add approximately one to 1.5 milliliters of substrate solution dropwise on the membrane. Immediately place another acetate sheet on it and incubate for five minutes at room temperature. Squeeze out the excess substrate solution before imaging.
Using a gel documentation imaging system, collect multiple unsaturated images at different time points for analysis. After southern blotting and hybridization, the telomere restriction fragments were clearly visible indicated by a smear.
